Methods and compositions for detecting esophageal neoplasias and/or metaplasias in the esophagus

ABSTRACT

The disclosure provides methods for identifying genomic loci (e.g., vimentin and/or SqBE18) that are differentially methylated in metaplasias (e.g., Barrett&#39;s esophagus) and/or neoplastic cancers (e.g., esophageal cancers). Identification of methylated genomic loci has numerous uses, including for example, to characterize disease risk, to predict responsiveness to therapy, to non-invasively diagnose subjects and to treat subjects determined to have gastrointestinal metaplasias and/or neoplasias.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 62/358,701, filed on Jul. 6, 2016. The disclosure of the foregoingapplication is hereby incorporated by reference in its entirety.

FUNDING

This invention was made with government support under UO1CA152756;U54CA163060; and P50CA150964 awarded by the National Institutes ofHealth (NIH). The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jul. 5, 2017, is named1848493_0002_098_WO1_SL.TXT, and is 14,247,735 bytes in size.

BACKGROUND

Over 15,000 new cases of esophageal cancer were diagnosed in 2010, andthere were nearly as many deaths from this cancer alone. As with othercancers, this rate can be decreased by improved methods for diagnosis.Although methods for detecting esophageal cancer exist, the methods arenot ideal. Generally, a combination of endoscopy, isolation of cells(for example, via collection of cells/tissues from a fluid sample orfrom a tissue sample), and/or imaging technologies are used to identifycancerous cells and tumors. While upper endoscopy, usually performed bya gastroenterologist, can detect neoplasias of the esophagus, as well asof the stomach and duodenum, it is an uncomfortable and expensiveprocedure. Other detection procedures, such as barium esophogography arealso available, but are associated with false positives, falsenegatives, and cost and discomfort issues.

Because of the disadvantages of existing methods for detecting ortreating esophageal neoplasias/cancers, new methods are needed foresophageal neoplasia/cancer diagnosis and therapy.

SUMMARY OF THE DISCLOSURE

In certain aspects, the present disclosure is based in part on thediscovery of particular human genomic DNA regions (also referred toherein as informative loci or patches) in which the cytosines within CpGdinucleotides are differentially methylated in esophageal neoplasiacompared to normal human tissues.

A first aspect of the present disclosure provides a method of diagnosingwhether a subject has an esophageal neoplasia or metaplasia, comprising:obtaining a sample from a subject; measuring the amount of methylatedcytosines in CpG dinucleotides in a vimentin nucleic acid sequence, orportion thereof, obtained from the sample; wherein if at least 80% ofthe cytosines in CpG dinucleotides in the vimentin nucleic acidsequence, or portion thereof, are methylated, than the vimentin nucleicacid sequence, or portion thereof, is considered a methylated read; andmeasuring the number of methylated reads present in the sample; whereinif at least 1% of the vimentin nucleic acid sequences, or portionsthereof, in the sample are methylated reads, than the subject isdetermined to have an esophageal neoplasia or metaplasia.

In some embodiments, the vimentin nucleic acid sequences from the sampleare treated with bisulfite. Optionally, the sequence of the bisulfiteconverted nucleic acid sequences is determined by next-generationsequencing. In some embodiments, the level of methylated cytosines isdetermined in an amplified portion of the vimentin nucleic acid sequenceobtained from the subject. Optionally, between the amplificationprimers, the amplified portion comprises 10 dinucleotides thatcorrespond to or are derived from 10 CpG dinucleotides present in thenative non-bisulfite treated vimentin genomic sequence. In someembodiments, the primers used to amplify the portion of the vimentinnucleic acid sequence comprise SEQ ID NOs: 16209 and 16210. Optionally,the amplified portion comprises the nucleotide sequence of SEQ ID NOs:16207 and/or 16208. In some embodiments, the 10 CpGs correspond to thosethat, after bisulfite treatment, are included in SEQ ID Nos: 16211 and16212.

In some embodiments, if at least 1.05% of the vimentin nucleic acidsequences, or portions thereof, in the sample are methylated reads, thanthe subject is determined to have an esophageal neoplasia or metaplasia.Optionally, if at least 3% of the vimentin nucleic acid sequences, orportions thereof, in the sample are methylated reads, than the subjectis determined to have an esophageal neoplasia or metaplasia. In someembodiments, if at least 5% of the vimentin nucleic acid sequences, orportions thereof, in the sample are methylated reads, than the subjectis determined to have an esophageal neoplasia or metaplasia.

In some embodiments, if the subject is determined to have an esophagealneoplasia or metaplasia, then administering to the subject cryotherpy,photodynamic therapy (PDT); radiofrequency ablation (RFA); laserablation, argon plasma coagulation (APC); electrocoagulation(electrofulguration); esophageal stent, surgery, and/or a therapeuticagent.

A second aspect of the present disclosure provides a method of treatinga subject having an esophageal neoplasia or metaplasia, wherein it hasbeen previously determined that at least 1% of the vimentin nucleic acidsequences, or portions thereof, in a sample from the subject have atleast 80% of the CpG dinucleotides methylated, wherein the methodcomprises administering to the subject cryotherpy, photodynamic therapy(PDT); radiofrequency ablation (RFA); laser ablation; argon plasmacoagulation (APC); electrocoagulation (electrofulguration); esophagealstent, surgery, and/or a therapeutic agent.

In some embodiments, the therapeutic agent is a proton pump inhibitor, aHistamine H2 receptor blocking agents, an anti-reflux medication, a drugthat moves food thru the gastrointestinal tract more quickly,carboplatin and paclitaxel (Taxol®) (which may be combined withradiation); cisplatin and 5-fluorouracil (5-FU) (often combined withradiation); ECF: epirubicine (Ellence®), cisplatin, and 5-FU (especiallyfor gastroesophageal junction tumors); DCF: docetaxel (Taxotere®),cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda®): oxaliplatinand either 5-FU or capecitabine: doxorubicin (Adriamycin®), bleomycin,mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan, andirinotecan (Camptosar®), trastuzumab, and/or ramucirumab. Optionally,the surgery is endoscopic mucosal resection (EMR), esophagectomy, and/oranti-reflux surgery.

In some embodiments, the disclosure provides for a method of diagnosingwhether a subject has an esophageal neoplasia or metaplasia, comprising:obtaining a sample from a subject by means of a brushing (e.g, acytology brushing); measuring the amount of methylated cytosines in CpGdinucleotides in an SqBE18 nucleic acid sequence, or portion thereof,obtained from the sample; wherein if at least 70% or at least 75% of thecytosines in CpG dinucleotides in the SqBE18 nucleic acid sequence, orportion thereof, are methylated, than the SqBE18 nucleic acid sequence,or portion thereof, is considered a methylated read; and measuring thenumber of methylated reads present in the sample; wherein if at least 3%of the SqBE18 nucleic acid sequences, or portions thereof, in the sampleare methylated reads, than the subject is determined to have anesophageal neoplasia or metaplasia. In some embodiments, the SqBE18nucleic acid sequences from the sample are treated with bisulfite. Insome embodiments, the sequence of the bisulfite converted nucleic acidsequences is determined by next-generation sequencing. In someembodiments, the level of methylated cytosines is determined in anamplified portion of the SqBE18 nucleic acid sequence obtained from thesubject. In some embodiments, the amplified portion comprises 21dinucleotides that correspond to or are derived from 21 CpGdinucleotides present in the native non-bisulfite treated SqBE18 genomicsequence. In some embodiments, if at least 3.11% of the SqBE18 nucleicacid sequences, or portions thereof, in the sample are methylated reads,than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if the subject is determined to have anesophageal neoplasia or metaplasia, then the method further comprisesadministering to the subject cryotherpy, photodynamic therapy (PDT);radiofrequency ablation (RFA); laser ablation; argon plasma coagulation(APC); electrocoagulation (electrofulguration); esophageal stent,surgery, and/or a therapeutic agent.

In some embodiments, the disclosure provides for a method of treating asubject having an esophageal neoplasia or metaplasia, wherein it hasbeen previously determined that at least 3% of the SqBE18 nucleic acidsequences, or portions thereof, in a brushing (e.g., a cytologybrushing) sample from the subject have at least 70% or at least 75% ofthe CpG dinucleotides methylated, wherein the method comprisesadministering to the subject cryotherpy, photodynamic therapy (PDT);radiofrequency ablation (RFA); laser ablation; argon plasma coagulation(APC); electrocoagulation (electrofulguration); esophageal stent,surgery, and/or a therapeutic agent. In some embodiments, thetherapeutic agent is a proton pump inhibitor, a Histamine H2 receptorblocking agents, an anti-reflux medication, a drug that moves food thruthe gastrointestinal tract more quickly, carboplatin and paclitaxel(Taxol®) (which may be combined with radiation); cisplatin and5-fluorouracil (5-FU) (often combined with radiation); ECF: epirubicine(Ellence®), cisplatin, and 5-FU (especially for gastroesophagealjunction tumors); DCF: docetaxel (Taxotere®), cisplatin, and 5-FU;Cisplatin with capecitabine (Xeloda®); oxaliplatin and either 5-FU orcapecitabine; doxorubicin (Adriamycin®), bleomycin, mitomycin,methotrexate, vinorelbine (Navelbine®), topotecan, and irinotecan(Camptosar®), trastuzumab, and/or ramucirumab. In some embodiments, thesurgery is endoscopic mucosal resection (EMR), esophagectomy, and/oranti-reflux surgery. In some embodiments, the disclosure provides for amethod of diagnosing whether a subject has an esophageal neoplasia ormetaplasia, comprising: obtaining a sample from a subject by means of aballoon; measuring the amount of methylated cytosines in CpGdinucleotides in an SqBE18 nucleic acid sequence, or portion thereof,obtained from the sample; wherein if at least 70% or at least 75% of thecytosines in CpG dinucleotides in the SqBE18 nucleic acid sequence, orportion thereof, are methylated, than the SqBE18 nucleic acid sequence,or portion thereof, is considered a methylated read; and measuring thenumber of methylated reads present in the sample; wherein if at least0.1% of the SqBE18 nucleic acid sequences, or portions thereof, in thesample are methylated reads, than the subject is determined to have anesophageal neoplasia or metaplasia. In some embodiments, the SqBE18nucleic acid sequences from the sample are treated with bisulfite. Insome embodiments, the sequence of the bisulfite converted nucleic acidsequences is determined by next-generation sequencing. In someembodiments, the level of methylated cytosines is determined in anamplified portion of the SqBE18 nucleic acid sequence obtained from thesubject. In some embodiments, the amplified portion comprises 21dinucleotides that correspond to or are derived from 21 CpGdinucleotides present in the native non-bisulfite treated SqBE18 genomicsequence. In some embodiments, if at least 0.76% of the SqBE18 nucleicacid sequences, or portions thereof, in the sample are methylated reads,than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if at least 1% of the SqBE18 nucleicacid sequences, or portions thereof, in the sample are methylated reads,than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if the subject is determined to have anesophageal neoplasia or metaplasia, then the method further comprisesadministering to the subject cryotherpy, photodynamic therapy (PDT),radiofrequency ablation (RFA); laser ablation; argon plasma coagulation(APC); electrocoagulation (electrofulguration); esophageal stent,surgery, and/or a therapeutic agent.

In some embodiments, the disclosure provides for a method of treating asubject having an esophageal neoplasia or metaplasia, wherein it hasbeen previously determined that at least 1% of the SqBE18 nucleic acidsequences, or portions thereof, in a balloon sample from the subjecthave at least 70% of the CpG dinucleotides methylated, wherein themethod comprises administering to the subject cryotherpy, photodynamictherapy (PDT); radiofrequency ablation (RFA); laser ablation; argonplasma coagulation (APC); electrocoagulation (electrofulguration);esophageal stent, surgery, and/or a therapeutic agent. In someembodiments, the therapeutic agent is a proton pump inhibitor, aHistamine H2 receptor blocking agents, an anti-reflux medication, a drugthat moves food thru the gastrointestinal tract more quickly,carboplatin and paclitaxel (Taxol®) (which may be combined withradiation); cisplatin and 5-fluorouracil (5-FU) (often combined withradiation); ECF: epirubicine (Ellence®), cisplatin, and 5-FU (especiallyfor gastroesophageal junction tumors); DCF: docetaxel (Taxotere®),cisplatin, and 5-FU: Cisplatin with capecitabine (Xeloda®); oxaliplatinand either 5-FU or capecitabine; doxorubicin (Adriamycin®), bleomycin,mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan, andirinotecan (Camptosar®), trastuzumab, and/or ramucirumab. In someembodiments, the surgery is endoscopic mucosal resection (EMR),esophagectomy, and/or anti-reflux surgery.

In some embodiments, the disclosure provides for a method of diagnosingwhether a subject has an esophageal neoplasia or metaplasia, comprising:obtaining a sample from a subject by means of a brushing (e.g., acytology brushing); measuring the amount of methylated cytosines in CpGdinucleotides in a vimentin nucleic acid sequence, or portion thereof,obtained from the sample; wherein if at least 80% of the cytosines inCpG dinucleotides in the vimentin nucleic acid sequence, or portionthereof, are methylated, than the vimentin nucleic acid sequence, orportion thereof, is considered a vimentin methylated read; measuring theamount of methylated cytosines in CpG dinucleotides in an SqBE18 nucleicacid sequence, or portion thereof, obtained from the sample, wherein ifat least 70% or 75% of the cytosines in CpG dinucleotides in the SqBE18nucleic acid sequence, or portion thereof, are methylated, than theSqBE18 nucleic acid sequence, or portion thereof, is considered anSqBE18 methylated read; and measuring the number of methylated readspresent in the sample; wherein if at least 1% of the vimentin nucleicacid sequences, or portions thereof, in the sample are vimentinmethylated reads, and wherein if at least 3% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are SqBE18 methylatedreads, than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, the vimentin and SqBE18 nucleic acidsequences from the sample are treated with bisulfite. In someembodiments, the sequence of the bisulfite converted nucleic acidsequences is determined by next-generation sequencing. In someembodiments, the level of methylated cytosines is determined in anamplified portion of the vimentin nucleic acid sequence and in anamplified portion of the SqBE18 nucleic acid sequence obtained from thesubject In some embodiments, the amplified portion of the SqBE18 nucleicacid sequence comprises 21 dinucleotides that correspond to or arederived from 21 CpG dinucleotides present in the native non-bisulfitetreated SqBE18 genomic sequence In some embodiments, the amplifiedportion of the vimentin nucleic acid sequence comprises 10 dinucleotidesthat correspond to or are derived from 10 CpG dinucleotides present inthe native non-bisulfite treated vimentin genomic sequence. In someembodiments, if at least 1% of the vimentin nucleic acid sequences, orportions thereof, in the sample are vimentin methylated reads, whereinif at least 3.11% of the SqBE18 nucleic acid sequences, or portionsthereof, in the sample are methylated reads, than the subject isdetermined to have an esophageal neoplasia or metaplasia. In someembodiments, if the subject is determined to have an esophagealneoplasia or metaplasia, then the method further comprises administeringto the subject cryotherpy, photodynamic therapy (PDT); radiofrequencyablation (RFA); laser ablation, argon plasma coagulation (APC);electrocoagulation (electrofulguration); esophageal stent, surgery,and/or a therapeutic agent.

In some embodiments, the disclosure provides for a method of treating asubject having an esophageal neoplasia or metaplasia, wherein it hasbeen previously determined that at least 1% of the vimentin nucleic acidsequences, or portions thereof, in a brushing (e.g., a cytologybrushing) sample from the subject have at least 80% of the CpGdinucleotides methylated, wherein it has been previously determined thatat least 3% of the SqBE18 nucleic acid sequences, or portions thereof,in a brushing (e.g., a cytology brushing) sample from the subject haveat least 75% of the CpG dinucleotides methylated, and wherein the methodcomprises administering to the subject cryotherpy, photodynamic therapy(PDT); radiofrequency ablation (RFA); laser ablation; argon plasmacoagulation (APC); electrocoagulation (electrofulguration); esophagealstent, surgery, and/or a therapeutic agent. In some embodiments, thetherapeutic agent is a proton pump inhibitor, a Histamine H2 receptorblocking agents, an anti-reflux medication, a drug that moves food thruthe gastrointestinal tract more quickly, carboplatin and paclitaxel(Taxol®) (which may be combined with radiation); cisplatin and5-fluorouracil (5-FU) (often combined with radiation); ECF: epirubicine(Ellence®), cisplatin, and 5-FU (especially for gastroesophagealjunction tumors); DCF: docetaxel (Taxotere®), cisplatin, and 5-FU;Cisplatin with capecitabine (Xeloda®); oxaliplatin and either 5-FU orcapecitabine; doxorubicin (Adriamycin®), bleomycin, mitomycin,methotrexate, vinorelbine (Navelbine®), topotecan, and irinotecan(Camptosar®), trastuzumab, and/or ramucirumab. In some embodiments, thesurgery is endoscopic mucosal resection (EMR), esophagectomy, and/oranti-reflux surgery. In some embodiments, method of diagnosing whether asubject has an esophageal neoplasia or metaplasia, comprising: obtaininga sample from a subject by means of a balloon; measuring the amount ofmethylated cytosines in CpG dinucleotides in a vimentin nucleic acidsequence, or portion thereof, obtained from the sample; wherein if atleast 80% of the cytosines in CpG dinucleotides in the vimentin nucleicacid sequence, or portion thereof, are methylated, than the vimentinnucleic acid sequence, or portion thereof, is considered a vimentinmethylated read; measuring the amount of methylated cytosines in CpGdinucleotides in an SqBE18 nucleic acid sequence, or portion thereof,obtained from the sample; wherein if at least 70% or at least 75% of thecytosines in CpG dinucleotides in the SqBE18 nucleic acid sequence, orportion thereof, are methylated, than the SqBE18 nucleic acid sequence,or portion thereof, is considered a SqBE18 methylated read; andmeasuring the number of methylated reads present in the sample; whereinif at least 0.95% of the vimentin nucleic acid sequences, or portionsthereof, in the sample are vimentin methylated reads, and wherein if atleast 0.1% of the SqBE18 nucleic acid sequences, or portions thereof, inthe sample are SqBE18 methylated reads, than the subject is determinedto have an esophageal neoplasia or metaplasia. In some embodiments, thevimentin and SqBE18 nucleic acid sequences from the sample are treatedwith bisulfite. In some embodiments, the sequence of the bisulfiteconverted nucleic acid sequences is determined by next-generationsequencing. In some embodiments, the level of methylated cytosines isdetermined in an amplified portion of the vimentin nucleic acid sequenceand in an amplified portion of the SqBE18 nucleic acid sequence obtainedfrom the subject. In some embodiments, the amplified portion comprises21 dinucleotides that correspond to or are derived from 21 CpGdinucleotides present in the native non-bisulfite treated SqBE18 genomicsequence. In some embodiments, the amplified portion of the vimentinnucleic acid sequence comprises 10 dinucleotides that correspond to orare derived from 10 CpG dinucleotides present in the nativenon-bisulfite treated vimentin genomic sequence. In some embodiments, ifat least 1% of the vimentin nucleic acid sequences, or portions thereofand if at least 0.76% of the SqBE18 nucleic acid sequences, or portionsthereof, in the sample are methylated reads, than the subject isdetermined to have an esophageal neoplasia or metaplasia. In someembodiments, if at least 1% of the vimentin nucleic acid sequences, orportions thereof and at least 1% of the SqBE18 nucleic acid sequences,or portions thereof, in the sample are methylated reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if the subject is determined to have an esophagealneoplasia or metaplasia, then the method further comprises administeringto the subject cryotherpy, photodynamic therapy (PDT), radiofrequencyablation (RFA); laser ablation; argon plasma coagulation (APC);electrocoagulation (electrofulguration); esophageal stent, surgery,and/or a therapeutic agent.

In some embodiments, the disclosure provides for a method of treating asubject having an esophageal neoplasia or metaplasia, wherein it hasbeen previously determined that at least 1% of the SqBE18 nucleic acidsequences, or portions thereof, in a balloon sample from the subjecthave at least 70% or at least 75% of the CpG dinucleotides methylated,wherein the method comprises administering to the subject cryotherpy,photodynamic therapy (PDT); radiofrequency ablation (RFA); laserablation; argon plasma coagulation (APC); electrocoagulation(electrofulguration); esophageal stent, surgery, and/or a therapeuticagent. In some embodiments, the therapeutic agent is a proton pumpinhibitor, a Histamine H2 receptor blocking agents, an anti-refluxmedication, a drug that moves food thru the gastrointestinal tract morequickly, carboplatin and paclitaxel (Taxol®) (which may be combined withradiation); cisplatin and 5-fluorouracil (5-FU) (often combined withradiation); ECF: epirubicine (Ellence®), cisplatin, and 5-FU (especiallyfor gastroesophageal junction tumors); DCF: docetaxel (Taxotere®),cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda®); oxaliplatinand either 5-FU or capecitabine; doxorubicin (Adriamycin®), bleomycin,mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan, andirinotecan (Camptosar®), trastuzumab, and/or ramucirumab. In someembodiments, the surgery is endoscopic mucosal resection (EMR),esophagectomy, and/or anti-reflux surgery.

In some embodiments, the primers used to amplify the portion of theSqBe18 nucleic acid sequence in any of the methods disclosed hereincomprise SEQ ID NOs: 8388 and/or 8402. In some embodiments, theamplified portion comprises the nucleotide sequence of SEQ ID NOs: 8318,8360, 8332 and/or 8374. In some embodiments, the amplified portioncomprises the nucleotide sequence of SEQ ID NOs: 8332 and/or 8374. Insome embodiments, the determination that the subject has an esophagealneoplasia or metaplasia is confirmed by an additional diagnostic assayIn some embodiments, the additional diagnostic assay is an endoscopicassay.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D show the Receiver Operating Characteristic (ROC) curves ofmethylated vimentin (VIM) and SqBE18 measurement in esophageal cytologybrushings of the normal appearing GE junction or of endoscopicallyvisualized BE or EAC. FIG. 1A shows a ROC curve based on Next-GenerationBisulfite Sequencing assay for SqBE18 in the training set of 61 controlsand 108 cases. FIG. 1B shows a ROC curve based on Next-GenerationBisulfite Sequencing VIM assay in the training set of 59 controls and107 cases.

FIG. 1C shows a ROC curve based on Next-Generation Bisulfite Sequencingassay for SqBE18 in the validation set of 28 controls and 115 cases.FIG. 1D shows a ROC curve based on Next-Generation Bisulfite SequencingVIM assay in the validation set of 27 controls and 117 cases. Area underthe curve (AUC), and the sensitivity and specificity of the assay at theoptimal cutpoint are listed for each of FIGS. 1A-1D. The numbers ofcases and controls for each marker in the training and validation setreflect the number of samples that were sequenced with the depth ofgreater than 84) reads for each marker. (median read depth was 3,809 forSqBE18, and 10,021 for VIM).

FIGS. 2A and 2B show VIM and SqBE18 performance at different numbers ofCpG cutoff for positivity, using the ROC cutoff for percent methylation.FIG. 2A shows a VIM Sensitivity and specificity curve. The 8+CpG cutoff(blue box), maximizes the sum of specificity for controls andsensitivity for cases. FIG. 2B shows a SqBE18 Sensitivity andspecificity curve. The 15+, 16+, and 17+CpG cutoffs offer identicalmaximum sensitivity+specificity sum for SqBE18, 16+CpGs (blue box), waschosen as the middle of this range.

FIG. 3 is a table showing the performance of VIM and SqBE18 in atraining set of brushings. Specificity Controls of the GastroesophagealJunction (GEJ)=Unaffected controls (individuals with GERD, erosiveesophagitis (EE), or no pathology detected during endoscopy (“other”));SSBE=short-segment Barrett's Esophagus (1 to 3 cm)); LSBE=Barrett'sEsophagus (3 cm or more); LGD=Barret's Esophagus with Low-GradeDysplasia; HGD=Barrett's Esophagus with High-Grade Dysplasia:Cancer=includes EAC (Esophageal adenocarcinoma) and JCA (Junctionalcancer of the esophagus).

FIG. 4 is a table showing the performance of VIM and SqBE18 inVALIDATION set of brushings. Specificity Controls of theGastroesophageal Junction (GEJ)=Unaffected controls (individuals withGERD, erosive esophagitis (EE), or no pathology detected duringendoscopy (“other”)); SSBE=short-segment Barrett's Esophagus (1 to 3cm)); LSBE=Barret's Esophagus (3 cm or more); LGD=Barret's Esophaguswith Low-Grade Dysplasia; HGD=Barrett's Esophagus with High-GradeDysplasia; Cancer=includes EAC (Esophageal adenocarcinoma) and JCA(Junctional cancer of the esophagus).

FIG. 5 is a table showing the performance of VIM and SqBE18 in acombination set of brushings. This table includes all the samples fromtraining and validation set of brushings combined. Specificity Controlsof the Gastroesophageal Junction (GEJ)=Unaffected controls (individualswith GERD, erosive esophagitis (EE), or no pathology detected duringendoscopy (“other”)), SSBE=short-segment Barrett's Esophagus (1 to 3cm)). LSBE=Barrett's Esophagus (3 cm or more), LGD=Barret's Esophaguswith Low-Grade Dysplasia; HGD=Barrett's Esophagus with High-GradeDysplasia; Cancer=includes EAC (Esophageal adenocarcinoma) and JCA(Junctional cancer of the esophagus).

FIGS. 6A and 6B show Receiver Operating Characteristic (ROC) curves ofmethylated VIM assayed on esophageal balloon samplings of the distalesophagus. FIG. 6A shows a ROC curve based on Next-Generation BisulfiteSequencing assay for VIM in the training set of 38 controls and 50cases. FIG. 6B shows a ROC curve based on Next-Generation BisulfiteSequencing SqBE18 assay in the training set of 38 controls and 50 cases.Area under the curve (AUC), and the sensitivity and specificity of theassay at the optimal cutpoint are listed for each graph.

FIG. 7 is a table showing performance of VIM and SqBE18 in Esophagealballoon samples. Specificity Controls of the Gastroesophageal Junction(GEJ)=Unaffected controls (individuals with GERD, erosive esophagitis(EE), or no pathology detected during endoscopy (“other”)),SSBE=short-segment Barrett's Esophagus (to 3 cm)). LSBE=Barrett'sEsophagus (3 cm or more); LGD=Barret's Esophagus with Low-GradeDysplasia; HGD=Barrett's Esophagus with High-Grade Dysplasia;Cancer=includes EAC (Esophageal adenocarcinoma) and JCA (Junctionalcancer of the esophagus).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention,suitable methods and materials are described below. The materials,methods and examples are illustrative only, and are not intended to belimiting. All publications, patents and other documents mentioned hereinare incorporated by reference in their entirety.

Each embodiment of the invention described herein may be taken alone orin combination with one or more other embodiments of the invention.

Throughout this specification, the word “comprise” or variations such as“comprises” or “comprising” will be understood to imply the inclusion ofa stated integer or groups of integers but not the exclusion of anyother integer or group of integers.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The terms “adenoma” is used herein to describe any precancerousneoplasia or benign tumor of epithelial tissue, for example, aprecancerous neoplasia of the gastrointestinal tract, pancreas, and/orthe bladder.

The term “blood-derived fraction” herein refers to a component orcomponents of whole blood. Whole blood comprises a liquid portion (i.e.,plasma) and a solid portion (i.e., blood cells). The liquid and solidportions of blood are each comprised of multiple components; e.g.,different proteins in plasma or different cell types in the solidportion. One of these components or a mixture of any of these componentsis a blood-derived fraction as long as such fraction is missing one ormore components found in whole blood.

The term “esophagus” is intended to encompass the upper portion of thedigestive system spanning from the back of the oral cavity, passingdownwards through the rear part of the mediastinum, through thediaphragm and into the stomach.

The term “esophageal cancer” is used herein to refer to any cancerousneoplasia of the esophagus.

“Barrett's esophagus” as used herein refers to an abnormal change(metaplasia) in the cells of the lower portion of the esophagus.Barrett's is characterized the finding of intestinal metaplasia in theesophagus.

A “brushing” of the esophagus, as referred to herein, may be obtainedusing any of the means known in the art. In some embodiments, a brushingis obtained by contacting the esophagus with a brush, a cytology brush,a sponge, a balloon, or with any other device or substance that contactsthe esophagus and obtains an esophageal sample.

“Cells,” “host cells” or “recombinant host cells” are terms usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The terms “compound”, “test compound,” “agent”, and “molecule” are usedherein interchangeably and are meant to include, but are not limited to,peptides, nucleic acids, carbohydrates, small organic molecules, naturalproduct extract libraries, and any other molecules (including, but notlimited to, chemicals, metals, and organometallic compounds).

The term “compound-converted DNA” herein refers to DNA that has beentreated or reacted with a chemical compound that converts unmethylated Cbases in DNA to a different nucleotide base. For example, one suchcompound is sodium bisulfite, which converts unmethylated C to U. If DNAthat contains conversion-sensitive cytosine is treated with sodiumbisulfite, the compound-converted DNA will contain U in place of C. Ifthe DNA which is treated with sodium bisulfite contains onlymethylcytosine, the compound-converted DNA will not contain uracil inplace of the methylcytosine.

The term “de-methylating agent” as used herein refers agents thatrestore activity and/or gene expression of target genes silenced bymethylation upon treatment with the agent. Examples of such agentsinclude without limitation 5-azacytidine and 5-aza-2′-deoxycytidine.

The term “detection” is used herein to refer to any process of observinga marker, or a change in a marker (such as for example the change in themethylation state of the marker), in a biological sample, whether or notthe marker or the change in the marker is actually detected. In otherwords, the act of probing a sample for a marker or a change in themarker, is a “detection” even if the marker is determined to be notpresent or below the level of sensitivity. Detection may be aquantitative, semi-quantitative or non-quantitative observation.

The term “differentially methylated nucleotide sequence” refers to aregion of a genomic loci that is found to be methylated in a in cancertissues or cell lines, but not methylated in the normal tissues or celllines.

The term “neoplasia” as used herein refers to an abnormal growth oftissue. As used herein, the term “neoplasia” may be used to refer tocancerous and non-cancerous tumors, as well as to Barrett's esophagus(which may also be referred to herein as a metaplasia) and Barrett'sesophagus with dysplasia In some embodiments, the Barrett's esophaguswith dysplasia is Barrett's esophagus with high grade dysplasia. In someembodiments, the Barrett's esophagus with dysplasia is Barrett'sesophagus with low grade dysplasia. In some embodiments, the neoplasiais a cancer (e.g., esophageal adenocarcinoma).

“Gastrointestinal neoplasia” refers to neoplasia of the upper and lowergastrointestinal tract. As commonly understood in the art, the uppergastrointestinal tract includes the esophagus, stomach, and duodenum;the lower gastrointestinal tract includes the remainder of the smallintestine and all of the large intestine.

The terms “healthy”, “normal,” and “non-neoplastic” are usedinterchangeably herein to refer to a subject or particular cell ortissue that is devoid (at least to the limit of detection) of a diseasecondition, such as a neoplasia.

“Homology” or nor “similarity” refers to sequence similarity between twopeptides or between two nucleic acid molecules. Homology and identitycan each be determined by comparing a position in each sequence whichmay be aligned for purposes of comparison. When an equivalent positionin the compared sequences is occupied by the same base or amino acid,then the molecules are identical at that position; when the equivalentsite occupied by the same or a similar amino acid residue (e.g., similarin steric and/or electronic nature), then the molecules can be referredto as homologous (similar) at that position. Expression as a percentageof homology/similarity or identity refers to a function of the number ofidentical or similar amino acids at positions shared by the comparedsequences. A sequence which is “unrelated or “non-homologous” shares, insome embodiments, less than 40% identity, and in particular embodiments,less than 25% identity with a sequence of the present invention. Incomparing two sequences, the absence of residues (amino acids or nucleicacids) or presence of extra residues also decreases the identity andhomology/similarity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid and protein sequences ofthe present invention may be used as a “query sequence” to perform asearch against public databases to, for example, identify other familymembers, related sequences or homologs. Such searches can be performedusing the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.(1990) J Mol. Biol. 215:403-10. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes. Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used. See www.ncbi.nlm.nih.gov.

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M, and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073,1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)). BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul. S., et al., NCBI NLM NIH Bethesda. Md. 20894: Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990)). The well known Smith Watermanalgorithm may also be used to determine identity.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to.”

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules in a form which does not occur innature. Moreover, an “isolated nucleic acid” is meant to include nucleicacid fragments which are not naturally occurring as fragments and wouldnot be found in the natural state.

The term “methylation-specific PCR” (“MSP”) herein refers to apolymerase chain reaction in which amplification of thecompound-converted template sequence is performed. Two sets of primersare designed for use in MSP. Each set of primers comprises a forwardprimer and a reverse primer. One set of primers, calledmethylation-specific primers (see below), will amplify thecompound-converted template sequence if C bases in CpG dinucleotideswithin the DNA are methylated. Another set of primers, calledunmethylation-specific primers or primers for unmethylated sequences andthe like (see below), will amplify the compound-converted templatesequences if C bases in CpG dinucleotides within the DNA are notmethylated.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA) The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single-stranded (such assense or antisense) and double-stranded polynucleotides.

“Operably linked” when describing the relationship between two DNAregions simply means that they are functionally related to each other.For example, a promoter or other transcriptional regulatory sequence isoperably linked to a coding sequence if it controls the transcription ofthe coding sequence.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

The terms “proteins” and “polypeptides” are used interchangeably herein.

A “sample” includes any material that is obtained or prepared fordetection of a molecular marker or a change in a molecular marker suchas for example the methylation state, or any material that is contactedwith a detection reagent or detection device for the purpose ofdetecting a molecular marker or a change in the molecular marker.

As used herein, “obtaining a sample” includes directly retrieving asample from a subject to be assayed, or directly retrieving a samplefrom a subject to be stored and assayed at a later time. Alternatively,a sample may be obtained via a second party. That is, a sample may beobtained via, e.g., shipment, from another individual who has retrievedthe sample, or otherwise obtained the sample.

A “subject” is any organism of interest, generally a mammalian subject,such as a mouse, and in particular embodiments, a human subject.

As used herein, the term “specifically hybridizes” refers to the abilityof a nucleic acid probe/primer of the invention to hybridize to at least12, 15, 20, 25, 30, 35, 40, 45, 50 or 100 consecutive nucleotides of atarget sequence, or a sequence complementary thereto, or naturallyoccurring mutants thereof, such that it has, in some embodiments, lessthan 15%, less than 10%, or less than 5% background hybridization to acellular nucleic acid (e.g., mRNA or genomic DNA) other than the targetgene. A variety of hybridization conditions may be used to detectspecific hybridization, and the stringency is determined primarily bythe wash stage of the hybridization assay. Generally high temperaturesand low salt concentrations give high stringency, while low temperaturesand high salt concentrations give low stringency. Low stringencyhybridization is achieved by washing in, for example, about 2.0×SSC at50° C., and high stringency is achieved with about 0.2×SSC at 50° C.Further descriptions of stringency are provided below.

As applied to polypeptides, the term “substantial sequence identity”means that two peptide sequences, when optimally aligned such as by theprograms GAP or BESTFIT using default gap, share at least 90 percentsequence identity, in some embodiments, at least 95 percent sequenceidentity, or at least 99 percent sequence identity or more. In someembodiments, residue positions which are not identical differ byconservative amino acid substitutions. For example, the substitution ofamino acids having similar chemical properties such as charge orpolarity is not likely to affect the properties of a protein. Examplesinclude glutamine for asparagine or glutamic acid for aspartic acid.

An “informative loci” as used herein, refers to any of the nucleic acidsequences disclosed herein that may have altered (e.g., increased)methylation in a sample (e.g., an esophageal tissue sample) from asubject having Barrett's esophagus and/or an esophageal neoplasia ascompared to the methylation patterns of the corresponding nucleic acidsequence in a sample from a healthy control subject. An example of aninformative loci is vimentin.

The term “Up3” as used herein refers to a nucleotide sequence comprisinga sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identity to the sequence of SEQ ID NO: 12563, 12581,12599, 12617, or fragments or reverse complements thereof. In someembodiments, the Up3 sequence refers to a bisulfite converted nucleotidesequence comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ IDNO: 12569, 12587, 12605 or 12623, or fragments or reverse complementsthereof. In some embodiments, the Up3 sequence refers to a bisulfiteconverted product of a methylated nucleotide sequence comprising asequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity to the sequence of SEQ ID NO: 12575, 12593, 12611or 12629, or fragments or reverse complements thereof. In someembodiments, the Up3 sequence may be amplified using primers comprisingthe sequence of SEQ ID NOs: 12635 and/or I2641, or fragments or reversecomplements thereof.

The term “Up10” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96% 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12564,12582, 12600 or 12618, or fragments or reverse complements thereof. Insome embodiments, the Up10 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 12570, 12588, 12606 or 12624, or fragments or reversecomplements thereof, in some embodiments, the Up10 sequence refers to abisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12576,12594, 12612 or 12630, or fragments or reverse complements thereof. Insome embodiments, the Up10 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 12636 and/or 12642, or fragmentsor reverse complements thereof.

The term “Up15-1” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12565,12583, 12601 or 12619, or fragments or reverse complements thereof. Insome embodiments, the Up15-1 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 12571, 12589, 12607 or 12625, or fragments or reversecomplements thereof. In some embodiments, the Up15-1 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12577,12595, 12613 or 12631, or fragments or reverse complements thereof. Insome embodiments, the Up15-1 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 12637 and/or 12643, or fragmentsor reverse complements thereof.

The term “Up15-2” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12565,12583, 12647 or 12656, or fragments or reverse complements thereof. Insome embodiments, the Up15-2 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 12571, 12589, 12650 or 12659, or fragments or reversecomplements thereof. In some embodiments, the Up15-2 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12577,12595, 12653 or 12662, or fragments or reverse complements thereof. Insome embodiments, the Up15-2 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 12665 and/or 12668, or fragmentsor reverse complements thereof.

The term “Up20-1” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12566,12584, 12602 or 12620, or fragments or reverse complements thereof. Insome embodiments, the Up20-1 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 12572, 12590, 12608 or 12626, or fragments or reversecomplements thereof. In some embodiments, the Up20-1 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90% 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12578,12596, 12614 or 12632, or fragments or reverse complements thereof. Insome embodiments, the Up20-1 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 12638 and/or 12644, or fragmentsor reverse complements thereof.

The term “Up20-2” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12566,12584, 12648 or 12657, or fragments or reverse complements thereof. Insome embodiments, the Up20-2 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 12572, 12590, 12651 or 12660, or fragments or reversecomplements thereof. In some embodiments, the Up20-2 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12578,12596, 12654 or 12663, or fragments or reverse complements thereof. Insome embodiments, the Up20-2 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 12666 and/or 12669, or fragmentsor reverse complements thereof.

The term “Up27” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%9%%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12567,12585, 12603 or 12621, or fragments or reverse complements thereof. Insome embodiments, the Up27 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 12573, 12591, 12609 or 12627, or fragments or reversecomplements thereof. In some embodiments, the Up27 sequence refers to abisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12579,12597, 12615 or 12633, or fragments or reverse complements thereof. Insome embodiments, the Up27 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 12639 and/or 12645, or fragmentsor reverse complements thereof.

The term “Up35-1” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12568,12586, 12604 or 12622, or fragments or reverse complements thereof. Insome embodiments, the Up35-1 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 12574, 12592, 12610 or 12628, or fragments or reversecomplements thereof. In some embodiments, the Up35-1 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12580,12598, 12616 or 12634, or fragments or reverse complements thereof. Insome embodiments, the Up35-1 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 12640 and/or 12646, or fragmentsor reverse complements thereof.

The term “Up35-2” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12568,12586, 12649 or 12658, or fragments or reverse complements thereof. Insome embodiments, the Up35-2 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 12574, 12592, 12652 or 12661, or fragments or reversecomplements thereof. In some embodiments, the Up35-2 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 12580,12598, 12655 or 12664, or fragments or reverse complements thereof. Insome embodiments, the Up35-2 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 12667 and/or 12670, or fragmentsor reverse complements thereof.

The term “SqBE 2” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8209,8251, 8293 or 8335, or fragments or reverse complements thereof. In someembodiments, the SqBE 2 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 950, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8223, 8265, 8307 or 8349, or fragments or reversecomplements thereof. In some embodiments, the SqBE 2 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8237,8279, 8321 or 8363, or fragments or reverse complements thereof. In someembodiments, the SqBE 2 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8377 and/or 8391, or fragments orreverse complements thereof.

The term “SqBE 5” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8210,8252, 8294 or 8336, or fragments or reverse complements thereof. In someembodiments, the SqBE 5 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8224, 8266, 8308 or 8350, or fragments or reversecomplements thereof. In some embodiments, the SqBE 5 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8238,8280, 8322 or 8364, or fragments or reverse complements thereof. In someembodiments, the SqBE 5 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8378 and/or 8392, or fragments orreverse complements thereof.

The term “SqBE 7” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8211,8253, 8295 or 8337, or fragments or reverse complements thereof. In someembodiments, the SqBE 7 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8225, 8267, 8309 or 8351, or fragments or reversecomplements thereof. In some embodiments, the SqBE 7 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8239,8281, 8323 or 8365, or fragments or reverse complements thereof. In someembodiments, the SqBE 7 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8379 and/or 8393, or fragments orreverse complements thereof.

The term “SqBE 9” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8212,8254, 8296 or 8338, or fragments or reverse complements thereof. In someembodiments, the SqBE 9 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8226, 8268, 8310 or 8352, or fragments or reversecomplements thereof. In some embodiments, the SqBE 9 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8240,8282, 8324 or 8366, or fragments or reverse complements thereof. In someembodiments, the SqBE 9 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8380 and/or 8394, or fragments orreverse complements thereof.

The term “SqBE 10” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,%%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8213,8255, 8297 or 8339, or fragments or reverse complements thereof. In someembodiments, the SqBE 10 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8227, 8269, 8311 or 8353, or fragments or reversecomplements thereof. In some embodiments, the SqBE 10 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8241,8283, 8325 or 8367, or fragments or reverse complements thereof. In someembodiments, the SqBE 10 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8381 and/or 8395, or fragments orreverse complements thereof.

The term “SqBE 11-1” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,%%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8214,8256, 8298 or 8340, or fragments or reverse complements thereof. In someembodiments, the SqBE 11-1 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8228, 8270, 8312 or 8354, or fragments or reversecomplements thereof. In some embodiments, the SqBE 11-1 sequence refersto a bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8242,8284, 8326 or 8368, or fragments or reverse complements thereof. In someembodiments, the SqBE 11-1 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8382 and/or 83%, or fragments orreverse complements thereof.

The term “SqBE 11-2” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8214,8256, 8405 or 8420, or fragments or reverse complements thereof. In someembodiments, the SqBE 11-2 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8228, 8270, 8410 or 8425, or fragments or reversecomplements thereof. In some embodiments, the SqBE 11-2 sequence refersto a bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8242,8284, 8415 or 8430, or fragments or reverse complements thereof. In someembodiments, the SqBE 11-2 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8435 and/or 8440, or fragments orreverse complements thereof.

The term “SqBE 13” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8215,8257, 8299 or 8341, or fragments or reverse complements thereof. In someembodiments, the SqBE 13 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8229, 8271, 8313 or 8355, or fragments or reversecomplements thereof. In some embodiments, the SqBE 13 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 940%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8243,8285, 8327 or 8369, or fragments or reverse complements thereof. In someembodiments, the SqBE 13 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8383 and/or 8397, or fragments orreverse complements thereof.

The term “SqBE 14-2” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8216,8258, 8406 or 8421, or fragments or reverse complements thereof. In someembodiments, the SqBE 14-2 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8230, 8272, 8411 or 8426, or fragments or reversecomplements thereof. In some embodiments, the SqBE 14-2 sequence refersto a bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8244,8286, 8416 or 8431, or fragments or reverse complements thereof. In someembodiments, the SqBE 14-2 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8436 and/or 8441, or fragments orreverse complements thereof.

The term “SqBE 15” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8217,8259, 8301 or 8343, or fragments or reverse complements thereof. In someembodiments, the SqBE 15 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8231, 8273, 8315 or 8357, or fragments or reversecomplements thereof. In some embodiments, the SqBE 15 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8245,8287, 8329 or 8371, or fragments or reverse complements thereof. In someembodiments, the SqBE 15 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8385 and/or 8399, or fragments orreverse complements thereof.

The term “SqBE 16-1” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,9%%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8218,8260, 8302 or 8344, or fragments or reverse complements thereof. In someembodiments, the SqBE 16-1 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8232, 8274, 8316 or 8358, or fragments or reversecomplements thereof. In some embodiments, the SqBE 16-1 sequence refersto a bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8246,8288, 8330 or 8372, or fragments or reverse complements thereof. In someembodiments, the SqBE 16-1 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8386 and/or 8400, or fragments orreverse complements thereof.

The term “SqBE 16-2” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8218,8260, 8407 or 8422, or fragments or reverse complements thereof. In someembodiments, the SqBE 16-2 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8232, 8274, 8412 or 8427, or fragments or reversecomplements thereof. In some embodiments, the SqBE 16-2 sequence refersto a bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8246,8288, 8417 or 8432, or fragments or reverse complements thereof. In someembodiments, the SqBE 16-2 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8437 and/or 8442, or fragments orreverse complements thereof.

The term “SqBE 17-1” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8219,8261, 8303 or 8345, or fragments or reverse complements thereof. In someembodiments, the SqBE 17-1 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8233, 8275, 8317 or 8359, or fragments or reversecomplements thereof. In some embodiments, the SqBE 17-1 sequence refersto a bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8247,8289, 8331 or 8373, or fragments or reverse complements thereof. In someembodiments, the SqBE 17-1 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8387 and/or 8401, or fragments orreverse complements thereof.

The term “SqBE18” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8220,8262, 8304 or 8346, or fragments or reverse complements thereof. In someembodiments, the SqBE18 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8234, 8276, 8318 or 8360, or fragments or reversecomplements thereof. In some embodiments, the SqBE18 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8248,8290, 8332 or 8374, or fragments or reverse complements thereof. In someembodiments, the SqBE18 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8388 and/or 8402, or fragments orreverse complements thereof.

The term “SqBE 22-1” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8221,8263, 8305 or 8347, or fragments or reverse complements thereof. In someembodiments, the SqBE 22-1 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8235, 8277, 8319 or 8361, or fragments or reversecomplements thereof. In some embodiments, the SqBE 22-1 sequence refersto a bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8249,8291, 8333 or 8375, or fragments or reverse complements thereof. In someembodiments, the SqBE 22-1 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8389 and/or 8403, or fragments orreverse complements thereof.

The term “SqBE 22-2” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8221,8263, 8409 or 8424, or fragments or reverse complements thereof. In someembodiments, the SqBE 22-2 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8235, 8277, 8414 or 8429, or fragments or reversecomplements thereof. In some embodiments, the SqBE 22-2 sequence refersto a bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8249,8291, 8419 or 8434, or fragments or reverse complements thereof. In someembodiments, the SqBE 22-2 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8439 and/or 8444, or fragments orreverse complements thereof.

The term “SqBE 23” as used herein refers to a nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8222,8264, 8306 or 8348, or fragments or reverse complements thereof. In someembodiments, the SqBE 23 sequence refers to a bisulfite convertednucleotide sequence comprising a sequence at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the sequenceof SEQ ID NO: 8236, 8278, 8320 or 8362, or fragments or reversecomplements thereof. In some embodiments, the SqBE 23 sequence refers toa bisulfite converted product of a methylated nucleotide sequencecomprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to the sequence of SEQ ID NO: 8250,8292, 8334 or 8376, or fragments or reverse complements thereof. In someembodiments, the SqBE 23 sequence may be amplified using primerscomprising the sequence of SEQ ID NOs: 8390 and/or 8404, or fragments orreverse complements thereof.

In some instances, any of the nucleotide sequences disclosed hereincontain one or more “Y” positions. Cytosine residues that may bemethylated or unmethylated, and hence may be bisulfite converted to T(if unmethylated) or remain as a C (if methylated), are designated witha “Y.” In some embodiments, a parental nucleotide sequence is fullyunmethylated if the sequence comprises a T at every Y position followingbisulfite conversion. In some embodiments, a parental nucleotidesequence is fully methylated if the sequence comprises a C at every Yposition following bisulfite conversion. In some embodiments, a parentalnucleotide sequence is partially methylated if the sequence comprises atleast one C at a Y position and at least one T at a Y position of thesequence following bisulfite conversion. In some embodiments, thebisulfite converted sequences disclosed herein comprise at least one Cat a Y position and at least one T at a Y position, i.e., the parentalsequence is partially methylated.

II. Overview

This disclosure is based at least in part on the recognition thatdifferential methylation of particular genomic loci (e.g., vimentinand/or SqBE18) may be indicative of a neoplasia or metaplasia of theupper gastrointestinal tract, e.g., esophagus. The present findingsdemonstrate that methylation at these genomic loci may be a usefulbiomarker of neoplasia in the upper gastrointestinal tract. The presentfindings further demonstrate that the status of methylation at thesegenomic loci used in combination with the status of somatic mutation(s)in TP53 may be a highly sensitive and specific biomarker of neoplasia inthe upper gastrointestinal tract.

In general, neoplasias may develop through one of at least threedifferent pathways, termed chromosomal instability, microsatelliteinstability, and the CpG island methylator phenotype (CIMP). Althoughthere is some overlap, these pathways tend to present somewhat differentbiological behavior. By understanding the pathway of tumor development,the target genes involved, and the mechanisms underlying the geneticinstability, it is possible to implement strategies to detect and treatthe different types of neoplasias.

This disclosure is based, at least in part, on the recognition thatcertain target genes may be silenced or inactivated by the differentialmethylation of CpG islands in the 5′ flanking or promoter regions of thetarget gene. CpG islands are clusters of cytosine-guanosine residues ina DNA sequence, which are prominently represented in the 5-flankingregion or promoter region of about half the genes in our genome. Inparticular, this application is based at least in part on therecognition that differential methylation of particular genomic loci maybe indicative of neoplasia of the upper gastrointestinal tractincluding, but not limited to, esophageal neoplasia.

Additionally, this disclosure is based, at least in part, on therecognition that somatic mutations in TP53 (e.g., any of the somaticTP53 mutations disclosed herein), in combination with methylation ofcertain informative loci as disclosed herein, may serve as usefulindicators of neoplasia, including esophageal neoplasia (e.g.,esophageal adenocarcinoma). In certain embodiments, the TP53 somaticmutation is any of the TP53 mutations disclosed herein In certainembodiments, the TP53 somatic mutation is any nonsynonymous somaticmutation known in the art. In certain embodiments, the TP53 somaticmutation is any one or more mutation at any one or more amino acidresidue corresponding to amino acid residue 72, 105, 108, 110, 113, 124,127, 132, 144, 152, 163, 175, 183, 194, 213, 214, 218, 232, 234, 248,265, 273, 278, 306, 337, 347, or 639 of SEQ ID NO: 16205. In certainembodiments, the TP53 somatic mutation is any one or more mutationselected from the group consisting of: Leu194Arg, Gly105Asp, Arg273His,Tyr163His, Ile232Thr, Arg213Ter, Arg273His, Arg248Gln, Arg175His, Arg10delinsGlnScr, Ser183Ter, Arg248Gln, Arg337Leu, Lys132Arg,Leu265ThrfsTer7, Arg306Ter, Cys124TrpfsTer25, Pro72Arg, Val218Glu,His214Leu, Gln144Ter, Phe113Ser, Tyr234His, Ser127Phe, Pro278Ala,Ala347Thr, and Pro152Leu of SEQ ID NO: 16205. In certain embodiments,the TP53 mutation is any one or more mutation at any one or morenucleotide position corresponding to nucleotide position 108, 215, 314,338, 380, 395, 430, 455, 487, 524, 548, 581, 637, 639, 641, 653, 695,700, 743, 818, 832, 916, 1010, or 1039 of SEQ ID NO: 16206.

The sequence of SEQ ID NO: 16205 (corresponding to GenBank Accession No.NP_000537.3) is as follows:

MEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVRVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALPNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD

The sequence of SEQ ID NO: 16206 (corresponding to GenBank Accession No.NM_000546.5) is as follows:

GATGGGATTGGGGTTTTCCCCTCCCATGTGCTCAAGACTGGCGCTAAAAGTTTTGAGCTTCTCAAAAGTCTAGAGCCACCGTCCAGGGAGCAGGTAGCTGCTGGGCTCCGGGGACACTTTGCGTTCGGGCTGGGAGCGTGCTTTCCACGACGGTGACACGCTTCCCTGGATTGGCAGCCAGACTGCCTTCCGGGTCACTGCCATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGGAAACATTTTCAGACCTATGGAAACTACTTCCTGAAAACAACGTTCTGTCCCCCTTGCCGTCCCAAGCAATGGATGATTTGATGCTGTCCCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCTCCCAGAATGCCAGAGGCTGCTCCCCCCGTGGCCCCTGCACCAGCAGCTCCTACACCGGCGGCCCCTGCACCAGCCCCCTCCTGGCCCCTGTCATCTTCTGTCCCTTCCCAGAAAACCTACCAGGGCAGCTACGGTTTCCGTCTGGGCTTCTTGCATTCTGGGACAGCCAAGTCTGTGACTTGCACGTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGCACTAAGCGAGCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGATCCGTGGGCGTGAGCGCTTCGAGATGTTCCGAGAGCTGAATGAGGCCTTGGAACTCAAGGATGCCCAGGCTGGGAAGGAGCCAGGGGGGAGCAGGGCTCACTCCAGCCACCTGAAGTCCAAAAAGGGTCAGTCTACCTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGGCCTGACTCAGACTGACATTCTCCACTTCTTGTTCCCCACTGACAGCCTCCCACCCCCATCTCTCCCTCCCCTGCCATTTTGGGTTTTGGGTCTTTGAACCCTTGCTTGCAATAGGTGTGCGTCAGAAGCACCCAGGACTTCCATTTGCTTTGTCCCGGGGCTCCACTGAACAAGTTGGCCTGCACTGGTGTTTTGTTGTGGGGAGGAGGATGGGGAGTAGGACATACCAGCTTAGATTTTAAGGTTTTTACTGTGAGGGATGTTTGGGAGATGTAAGAAATGTTCTTGCAGTTAAGGGTTAGTTTACAATCAGCCACATTCTAGGTAGGGGCCCACTTCACCGTACTAACCAGGGAAGCTGTCCCTCACTGTTGAATTTTCTCTAACTTCAAGGCCCATATCTGTGAAATGCTGGCATTTGCACCTACCTCACAGAGTGCATTGTGAGGGTTAATGAAATAATGTACATCTGGCCTTGAAACCACCTTTTATTACATGGGGTCTAGAACTTGACCCCCTTGAGGGTGCTTGTTCCCTCTCCCTGTTGGTCGGTGGGTTGGTAGTTTCTACAGTTGGGCAGCTGGTTAGGTAGAGGGAGTTGTCAAGTCTCTGCTGGCCCAGCCAAACCCTGTCTGACAACCTCTTGGTGAACCTTAGTACCTAAAAGGAAATCTCACCCCATCCCACACCCTGGAGGATTTCATCTCTTGTATATGATGATCTGGATCCACCAAGACTTGTTTTATGCTCAGGGTCAATTTCTTTTTTCTTTTTTTTTTTTTTTTTTCTTTTTCTTTGAGACTGGGTCTCGCTTTGTTGCCCAGGCTGGAGTGGAGTGGCGTGATCTTGGCTTACTGCAGCCTTTGCCTCCCCGGCTCGAGCAGTCCTGCCTCAGCCTCCGGAGTAGCTGGGACCACAGGTTCATGCCACCATGGCCAGCCAACTTTTGCATGTTTTGTAGAGATGGGGTCTCACAGTGTTGCCCAGGCTGGTCTCAAACTCCTGGGCTCAGGCGATCCACCTGTCTCAGCCTCCCAGAGTGCTGGGATTACAATTGTGAGCCACCACGTCCAGCTGGAAGGGTCAACATCTTTTACATTCTGCAAGCACATCTGCATTTTCACCCCACCCTTCCCCTCCTTCTCCCTTTTTATATCCCATTTTTATATCGATCTCTTATTTTACAATAAAACTTTGCTGCCACCTGTGTGTCTGAGGGGTG.

Esophageal adenocarcinoma (EAC) has steadily increased in incidence overrecent decades. With an 85% mortality rate this cancer is the mostrapidly increasing cause of cancer mortality from solid tumors in theAmerican population. There has thus been substantial interest indevelopment of screening approaches for early detection of EAC and itsprecursor lesions of Barrett's esophagus (BE). However, the majority ofEACs develop m patients without prior symptoms, and current approachesof endoscopic screening of individuals with persistent symptoms ofgastro-esophageal reflux disease, combined with longitudinal screeningof those found to have BE, have accordingly not had significant impacton reducing deaths from EACs.

As noted above, early detection of gastrointestinal neoplasia (e.g.,neoplasia of the upper gastrointestinal tract) coupled with appropriateintervention, is important for increasing patient survival rates.Present systems for screening for esophageal neoplasia are deficient fora variety of reasons, including a lack of specificity and/or sensitivity(e.g., barium swallow) or a high cost and intensive use of medicalresources (e.g., upper endoscopy or CT scan). Alternative systems fordetection of esophageal neoplasia would be useful in a wide range ofother clinical circumstances as well. For example, detecting esophagealneoplasia may select the patient to undergo therapies that include, butare not limited, to resection of the neoplasia (via endoscopic resectionor surgical resection), ablation of the neoplasia, chemotherapy, orradiation therapy. As a further example, patients who have receivedsurgical and/or pharmaceutical therapy for esophageal cancer mayexperience a relapse. It would be advantageous to have an alternativesystem for determining whether such patients have a recurrent orrelapsed neoplasia of the upper gastrointestinal tract. As a furtherexample, an alternative diagnostic system would facilitate monitoring anincrease, decrease or persistence of neoplasia of the uppergastrointestinal tract in a patient known to have such a neoplasia. Apatient undergoing chemotherapy may be monitored to assess theeffectiveness of the therapy.

III. Methylation of Informative Loci as Disease Biomarkers

The present disclosure relates at least in part to the identification ofgenomic loci whose altered DNA methylation is indicative of the presenceof esophageal neoplasias and/or metaplasias that include Barrett'sesophagus (BE) and/or esophageal adenocarcinoma (EAC). In someembodiments, the Barrett's esophagus is associated with dysplasia. Insome embodiments, the dysplasia is high-grade dysplasia In someembodiments, the dysplasia is low-grade dysplasia. In some embodiments,the methylation patterns of the informative loci as disclosed herein aredetermined in a sample taken from a subject as described herein and maybe used to distinguish between subjects having Barrett's esophagus andsubjects having high grade dysplasia and/or low grade dysplasia and/oresophageal adenocarcinoma. Examples of the informative loci are providedherein.

In some embodiments, any of the nucleotide sequences disclosed herein,or fragments or reverse complements thereof, may contain one or more “Y”residues. Cytosine residues that may be methylated or unmethylated, andhence may be bisulfite converted to T (if unmethylated) or remain as a C(if methylated), are designated with a “Y.” In some embodiments, one ormore of the Y residues in any of the sequences disclosed herein (orfragments or reverse complements thereof) designates a methylated C. Insome embodiments, one or more of the Y residues in any of the sequencesdisclosed herein (or fragments or reverse complements thereof)designates an unmethylated C. In some embodiments, at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 of the Y residues in any of the sequencesdisclosed herein (or fragments or reverse complements thereof)correspond to methylated C residues. In some embodiments, at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 of the Y residues in any of thesequences disclosed herein (or fragments or reverse complements thereof)correspond to unmethylated C residues. In some embodiments, at least10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the Y residuesin any of the sequences disclosed herein (or fragments or reversecomplements thereof) correspond to methylated C residues. In someembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% of the Y residues in any of the sequences disclosed herein (orfragments or reverse complements thereof) are correspond to unmethylatedC residues. In some embodiments, any of the sequences disclosed herein(or fragments or reverse complements thereof) is bisulfite-converted. Insome embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ofthe Y residues in any of the bisulfite-converted sequences disclosedherein (or fragments or reverse complements thereof) correspond to C. Insome embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 ofthe Y residues in any of the bisulfite-converted sequences disclosedherein (or fragments or reverse complements thereof) correspond to T. Insome embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 100% of the Y residues in any of the bisulfite-converted sequencesdisclosed herein (or fragments or reverse complements thereof)correspond to C residues. In some embodiments, at least 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, or 100% of the Y residues in any of thebisulfite-converted sequences disclosed herein (or fragments or reversecomplements thereof) correspond to T residues.

In some embodiments, an informative loci in a subject is considered“methylated” for the purposes of determining whether or not the subjectis prone to developing and/or has developed a metaplasia in theesophagus (e.g., Barrett's esophagus) or neoplasia (e.g., Barrett'sesophagus with dysplasia such as high-grade or low-grade dysplasia)(e.g., esophageal cancer such as esophageal adenocarcinoma) if the lociis at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%methylated. In some embodiments, a DNA sample from a subject is treatedwith bisulfite, and the resulting bisulfite sequence corresponds to anyof the nucleotide sequences disclosed herein comprising a “Y”nucleotide.

In some embodiments, if at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 of the Y residues of the bisulfite-converted sequence have a C, thesequence is considered “methylated” for the purposes of determiningwhether or not the subject is prone to developing and/or has developed ametaplasia in the esophagus (e.g., Barrett's esophagus) or neoplasia(e.g., Barrett's esophagus with dysplasia such as high-grade orlow-grade dysplasia) (e.g., esophageal cancer such as esophagealadenocarcinoma). In some embodiments, a DNA sample from a subject istreated with bisulfite, and the resulting bisulfite sequence correspondsto any of the nucleotide sequences disclosed herein comprising a “Y”nucleotide. In some embodiments, if at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% of the Y residues of the bisulfite-convertedsequence have a C, the sequence is considered “methylated” for thepurposes of determining whether or not the subject is prone todeveloping and/or has developed a metaplasia in the esophagus (e.g.,Barrett's esophagus) or neoplasia (e.g., Barrett's esophagus withdysplasia such as high-grade or low-grade dysplasia) (e.g., esophagealcancer such as esophageal adenocarcinoma). The disclosure provides forinformative loci that may be used to assess whether a subject (e.g, ahuman) has or is prone to developing a metaplasia in the esophagus(e.g., Barrett's esophagus) or neoplasia (e.g., Barrett's esophagus withdysplasia such as high-grade or low-grade dysplasia) (e.g., esophagealcancer such as esophageal adenocarcinoma). In some embodiments, one ormore informative loci, as defined herein, may be used for determiningwhether a subject has or is likely to develop, a metaplasia (e.g.,Barrett's esophagus). In some embodiments, one or more informative loci,as defined herein, may be used for determining whether a subject has oris likely to develop, a neoplasia (e.g., Barrett's esophagus with highgrade dysplasia, or an esophageal cancer such as esophagealadenocarcinoma). In some embodiments, one or more informative loci, asdefined herein, may be used to distinguish between whether a subject hasa metaplasia in the esophagus (e.g., Barrett's esophagus) or anesophageal neoplasia (e.g., Barrett's esophagus with high gradedysplasia, or an esophageal cancer such as esophageal adenocarcinoma).

In some embodiments, the informative loci include sequences associatedwith any one or more of the plus strand DNA sequences having at least80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to any of SEQ ID NOs: 1-428, 2569-2996, 5137-5531, 7507-7532,7663-7668, 7819-7842, 7963-7976, 8047-8060, 8131-8143, 8209-8222,8293-8306, 8405-8409, 8447-8632, 9563-9748, 10679-10825, 11561-11611,11867-11917, 12173-12219, 12455-12460, 12491-12496, 12527-12532,12563-12568, 12599-12604, 12647-12649, 12671-12907, 14093-14329,15515-15537, 15653-15692, 15893-15932, 16133-16137, 16163-16165,16181-16183, or 16199, or fragments or complements thereof. Inparticular embodiments, the informative loci include sequencesassociated with any one or more of the plus strand DNA sequences havingat least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identity to any of SEQ ID NOs: 7963-7976, 8047-8060, 8131-8143,12455-12460, 12491-12496, 12527-12532, 16163-16165, 16181-16183, or16199, or fragments or complements thereof. In some embodiments, theinformative loci are associated with increased methylation in bothBarrett's esophagus and esophageal adenocarcinoma samples, as comparedto the same sample types taken from a healthy control subject. In someembodiments, the informative loci that are associated with increasedmethylation in both Barrett's esophagus and esophageal adenocarcinomasamples include sequences associated with any one or more of the plusstrand DNA sequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs:1-428, 2569-2996, 5137-5531, 7507-7532, 7663-7668, 7819-7842, 7963-7976,8047-8060, 8131-8143, 8209-8222, 8293-8306, or 8405-8409, or fragmentsor complements thereof. In particular embodiments, the informative locithat are associated with increased methylation in both Barrett'sesophagus and esophageal adenocarcinoma samples include sequencesassociated with any one or more of the plus strand DNA sequences havingat least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identity to any of SEQ ID NOs: 7963-7976, 8047-8060, or8131-8143, or fragments or complements thereof. In some embodiments, theinformative loci are associated with increased methylation in anesophageal adenocarcinoma sample and/or a Barrett's with low grade orhigh grade dysplasia as compared to a sample of the same type taken froma subject having Barrett's esophagus without dysplasia. In someembodiments, the informative loci that are associated with increasedmethylation in an esophageal adenocarcinoma sample or a Barrett's withlow grade or high grade dysplasia sample include any one or more of thesequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs: 8447-8632,9563-9748, 10679-10825, 11561-11611, 11867-11917, 12173-12219,12455-12460, 12491-12496, or 12527-12532, or fragments or complementsthereof. In particular embodiments, the informative loci that areassociated with increased methylation in Barrett's with low gradedysplasia sample include any one or more of the sequences having atleast 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity to any of SEQ ID NOs: 12455-12460, 12491-12496, or12527-12532, or fragments or complements thereof. In particularembodiments, the informative loci that are associated with increasedmethylation in an esophageal adenocarcinoma sample or a Barrett's withhigh grade dysplasia sample include any one or more of the sequenceshaving at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identity to any of SEQ ID NOs: 12455-12460,12491-12496, or 12527-12532, or fragments or complements thereof. Insome embodiments, the informative loci are associated with reducedmethylation in an esophageal adenocarcinoma sample as compared to asample of the same type taken from a subject having Barrett's esophagus.In some embodiments, the informative loci that are associated withreduced methylation in an esophageal adenocarcinoma sample include anyone or more of the sequences having at least 80%, 85%, 87%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of SEQ IDNOs: 12671-12908, 14093-14329, 15515-15537, 15653-15692, 15893-15932,16133-16137, 16163-16165, 16181-16183, or 16199, or fragments orcomplements thereof. In particular embodiments, the informative locithat are associated with reduced methylation in an esophagealadenocarcinoma sample include any one or more of the sequences having atleast 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity to any of SEQ ID NOs: 16163-16165, 16181-16183, or 16199,or fragments or complements thereof.

In some embodiments, the informative loci or amplicon of the informativeloci are treated with an agent, such as bisulfite. In some embodiments,the informative loci include sequences that have been treated withbisulfite. In some embodiments, the disclosure provides for bisulfitecontrol sequences of any of the plus DNA strands disclosed herein. Insome embodiments, the disclosure provides for bisulfite-treatedunmethylated sequences of any of the plus DNA strands disclosed herein.In some embodiments, the bisulfite-converted plus-strand control DNAsequences include any one or more having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any ofSEQ ID NOs: 857-1284, 3425-3852, 5927-6321, 7559-7584, 7715-7740,7867-7890, 7991-8004, 8075-8088, 8157-8169, 8223-8236, 8307-8320,8410-8414, 8819-9004, 9935-10120, 10973-11119, 11663-11713, 11969-12019,12267-12313, 12467-12472, 12503-12508, or 12539-12544, 12569-12574,12605-12610, 12650-12652, or fragments or complements thereof. Inparticular embodiments, the bisulfite-converted plus-strand control DNAsequences include any one or more having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any ofSEQ ID NOs: 7991-8004, 8075-8088, 8157-8169, 8223-8236, 8307-8320,8410-8414, 12467-12472, 12503-12508, or 12539-12544, 12569-12574,12605-12610, 12650-12652, or fragments or complements thereof. In someembodiments, the informative loci are associated with increasedmethylation in both Barrett's esophagus and esophageal adenocarcinomasamples, as compared to the same sample types taken from a healthycontrol subject. In some embodiments, the disclosure provides forbisulfite-treated unmethylated sequences of any of the plus DNA strandsthat are associated with increased methylation in both Barrett'sesophagus and esophageal adenocarcinoma samples, as compared to the samesample types taken from a healthy control subject. In some embodiments,the bisulfite converted sequences of any of the plus DNA strands thatare associated with increased methylation in both Barrett's esophagusand esophageal adenocarcinoma samples are selected from the groupconsisting of sequences having at least 80%, 85%, 87%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs:857-1284, 3425-3852, 5927-6321, 7559-7584, 7715-7740, 7867-7890,7991-8004, 8075-8088, 8157-8169, 8223-8236, 8307-8320, or 8410-8414, orfragments or complements thereof. In particular embodiments, thebisulfite converted sequences of any of the plus DNA strands that areassociated with increased methylation in both Barrett's esophagus andesophageal adenocarcinoma samples include any one or morebisulfite-converted methylated plus-strand DNA sequences selected fromthe group consisting of sequences having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of:SEQ ID NOs: 7991-8004, 8075-8088, 8157-8169, 8223-8236, 8307-8320, or8410-8414, or fragments or complements thereof. In some embodiments, thedisclosure provides for bisulfite-treated unmethylated sequences of anyof the plus DNA strands that are associated with increased methylationin an esophageal adenocarcinoma sample or a Barrett's with low grade orhigh grade dysplasia sample as compared to a sample of the same typetaken from a subject having Barrett's esophagus without dysplasia. Insome embodiments, the bisulfite converted sequences of any of the plusDNA strands that are associated with increased methylation in anesophageal adenocarcinoma sample or a Barrett's with low grade or highgrade dysplasia sample include any one or more bisulfite-convertedmethylated plus-strand DNA sequences selected from the group consistinghaving at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identity to any of SEQ ID NOs: 8819-9004, 9935-10120,10973-11119, 11663-11713, 11969-12019, 12267-12313, 12467-12472,12503-12508, 12539-12544, 12569-12574, 12605-12610, or 12650-12652, orfragments or complements thereof. In particular embodiments, thebisulfite converted sequences of any of the plus DNA strands that areassociated with increased methylation in an esophageal adenocarcinomasample or a Barrett's with low grade or high grade dysplasia sampleinclude any one or more of the sequences having at least 80%, 85%, 87%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyof SEQ ID NOs: 12467-12472, 12503-12508, 12539-12544, 12569-12574,12605-12610, or 12650-12652, or fragments or complements thereof. Insome embodiments, the informative loci are associated with reducedmethylation in an esophageal adenocarcinoma sample as compared to asample of the same type taken from a subject having Barrett's esophagus.In some embodiments, the disclosure provides for methylated controlsequences of the plus DNA strand that are associated with reducedmethylation in an esophageal adenocarcinoma sample as compared to asample of the same type taken from a subject having Barrett's esophagus.In some embodiments, the methylated control sequences of any of the plusDNA strands that are associated with reduced methylation in anesophageal adenocarcinoma sample include any one or morebisulfite-converted methylated plus-strand DNA sequences selected fromthe group consisting having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs:13145-13381, 14567-14803, 15561-15583, 15733-15772, 15973-16012,16143-16147, 16169-16171, 16187-16189 or 16201, or fragments orcomplements thereof. In particular embodiments, the methylated controlsequences of any of the plus DNA strands that are associated withreduced methylation in an esophageal adenocarcinoma sample include anyone or more bisulfite-converted methylated plus-strand DNA sequencesselected from the group consisting having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any ofSEQ ID NOs: 16169-16171, 16187-16189 or 16201, or fragments orcomplements thereof.

In some embodiments, the informative loci or amplicon of the informativeloci are treated with an agent, such as bisulfite. In some embodiments,the informative loci include sequences that have been treated withbisulfite. In some embodiments, the informative loci include methylatednucleic acid sequences that have been treated with bisulfite. In someembodiments, the bisulfite-converted methylated plus-strand DNAsequences have at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs: 1285-1712,3853-4280, 6322-6716, 7585-7610, 7741-7766, 7891-7914, 8005-8018,8089-8102, 8170-8182, 8237-8250, 8321-8334, 8415-8419, 9005-9190,10121-10306, 11120-11266, 11714-11764, 12020-12070, 12314-12360,12473-12478, 12509-12514 or 12545-12550, 12575-12580, 12611-12616,12653-12655, or fragments or complements thereof. In particularembodiments, the bisulfite-converted methylated plus-strand DNAsequences have at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs: 8005-8018,8089-8102, 8170-8182, 12473-12478, 12509-12514 or 12545-12550, orfragments or complements thereof. In some embodiments, the informativeloci are associated with increased methylation in both Barrett'sesophagus and esophageal adenocarcinoma samples, as compared to the samesample types taken from a healthy control subject. In some embodiments,the informative loci that are associated with increased methylation inboth Barrett's esophagus and esophageal adenocarcinoma samples includeany one or more bisulfite-converted methylated plus-strand DNA sequencesselected from the group consisting of sequences having at least 80%,85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to any of SEQ ID NOs: 1285-1712, 3853-4280, 6322-6716,7585-7610, 7741-7766, 7891-7914, 8005-8018, 8089-8102, 8170-8182,8237-8250, 8321-8334, or 8415-8419, or fragments or complements thereof.In particular embodiments, the informative loci that are associated withincreased methylation in both Barrett's esophagus and esophagealadenocarcinoma samples include any one or more bisulfite-convertedmethylated plus-strand DNA sequences selected from the group consistingof sequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity to: SEQ ID NOs: 8005-8018,8089-8102, 8170-8182, 8237-8250, 8321-8334, or 8415-8419, or fragmentsor complements thereof. In some embodiments, the informative loci areassociated with increased methylation in an esophageal adenocarcinomasample or a Barrett's with low grade or high grade dysplasia sample ascompared to a sample of the same type taken from a subject havingBarrett's esophagus without dysplasia. In some embodiments, theinformative loci that are associated with increased methylation in anesophageal adenocarcinoma sample or a Barrett's with low grade or highgrade dysplasia sample include any one or more bisulfite-convertedmethylated plus-strand DNA sequences selected from the group consistingof sequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 9005-9190,10121-10306, 11120-11266, 11714-11764, 12020-12070, 12314-12360,12473-12478, 12509-12514, 12545-12550, 12575-12580, 12611-12616, or12653-12655, or fragments or complements thereof. In particularembodiments, the informative loci that are associated with increasedmethylation in an esophageal adenocarcinoma sample or a Barrett's withlow grade or high grade dysplasia sample include any one or more of thesequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 12473-12478,12509-12514, 12545-12550, 12575-12580, 12611-12616, or 12653-12655, orfragments or complements thereof. In some embodiments, the informativeloci are associated with reduced methylation in an esophagealadenocarcinoma sample as compared to a sample of the same type takenfrom a subject having Barrett's esophagus. In some embodiments, theinformative loci that are associated with reduced methylation in anesophageal adenocarcinoma sample include any one or morebisulfite-converted methylated plus-strand DNA sequences selected fromthe group consisting of sequences having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ IDNOs: 13382-13618, 14804-15040, 15584-15606, 15773-15812, 16013-16052,16148-16152, 16172-16174, 16190-16192 or 16202. In particularembodiments, the informative loci that are associated with reducedmethylation in an esophageal adenocarcinoma sample include any one ormore of the sequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs:16172-16174, 16190-16192 or 16202, or fragments or complements thereof.

In some embodiments, the informative loci include sequences associatedwith any of the minus strand DNA sequences having at least 80%, 85%,87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identityto any of SEQ ID NOs: 429-856, 2997-3424, 5532-5926, 7533-7558,7689-7714, 7843-7866, 7977-7990, 8061-8074, 8144-8156, 8251-8264,8335-8348, 8420-8424, 8633-8818, 9749-9934, 10826-10972, 11612-11662,11918-11968, 12220-12266, 12461-12466, 12497-12502, 12533-12538,12581-12586, 12617-12622, 12656-12658, 12909-13144, 14330-14566,15538-15560, 15693-15732, 15933-15972, 16138-16142, 16166-16168,16184-16186 or 16200, or fragments or complements thereof. In particularembodiments, the informative loci include sequences associated with anyof the minus strand DNA sequences having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any ofSEQ ID NOs: 7977-7990, 8061-8074, 8144-8156, 8251-8264, 8335-8348,8420-8424, 12461-12466, 12497-12502, 12533-12538, 12581-12586,12617-12622, 12656-12658, 16166-16168, 16184-16186 or 16200, orfragments or complements thereof. In some embodiments, the informativeloci are associated with increased methylation in both Barrett'sesophagus and esophageal adenocarcinoma samples, as compared to the samesample types taken from a healthy control subject. In some embodiments,the informative loci that are associated with increased methylation inboth Barrett's esophagus and esophageal adenocarcinoma samples includesequences associated with any one or more of the minus strand DNAsequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs: 429-856,2997-3424, 5532-5926, 7533-7558, 7689-7714, 7843-7866, 7977-7990,8061-8074, 8144-8156, 8251-8264, 8335-8348, 8420-8424, or fragments orcomplements thereof. In particular embodiments, the informative locithat are associated with increased methylation in both Barrett'sesophagus and esophageal adenocarcinoma samples include sequencesassociated with any one or more of the plus strand DNA sequences havingat least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identity to any of SEQ ID NOs: 8251-8264, 8335-8348. 8420-8424,or fragments or complements thereof. In some embodiments, theinformative loci are associated with increased methylation in anesophageal adenocarcinoma sample or a Barrett's with low grade or highgrade dysplasia sample as compared to a sample of the same type takenfrom a subject having Barrett's esophagus without dysplasia. In someembodiments, the informative loci that are associated with increasedmethylation in an esophageal adenocarcinoma sample or a Barrett's withlow grade or high grade dysplasia sample include any one or more of thesequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs: 8633-8818,9749-9934, 10826-10972, 11612-11662, 11918-11968, 12220-12266,12461-12466, 12497-12502, 12533-12538, 12581-12586, 12617-12622, or12656-12658, or fragments or complements thereof. In particularembodiments, the informative loci that are associated with increasedmethylation in an esophageal adenocarcinoma sample or a Barrett's withlow grade or high grade dysplasia sample include any one or more of thesequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs: 12461-12466,12497-12502, 12533-12538, 12581-12586, 12617-12622, or 12656-12658, orfragments or complements thereof. In some embodiments, the informativeloci are associated with reduced methylation in an esophagealadenocarcinoma sample as compared to a sample of the same type takenfrom a subject having Barrett's esophagus. In some embodiments, theinformative loci that are associated with reduced methylation in anesophageal adenocarcinoma sample include any one or more of thesequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to any of SEQ ID NOs: 12909-13144,14330-14566, 15538-15560, 15693-15732, 15933-15972, 16138-16142,16166-16168, 16184-16186 or 16200, or fragments or complements thereof.In particular embodiments, the informative loci that are associated withreduced methylation in an esophageal adenocarcinoma sample include anyone or more of the sequences having at least 80%, 85%, 87%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of SEQ IDNOs: 16166-16168, 16184-16186 or 16200, or fragments or complementsthereof.

In some embodiments, the informative loci or amplicon of the informativeloci are treated with an agent, such as bisulfite. In some embodiments,the informative loci include sequences that have been treated withbisulfite. In some embodiments, the disclosure provides for bisulfitecontrol sequences of any of the minus DNA strands disclosed herein. Insome embodiments, the disclosure provides for bisulfite-treatedsequences of any of the minus DNA strands disclosed herein. In someembodiments, the bisulfite-converted minus-strand control DNA sequencesinclude any one or more of the sequences having at least 80%, 85%, 87%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyof SEQ ID NOs: 1713-2140, 4281-4708, 6717-7111, 7611-7636, 7767-7792,7915-7938, 8019-8032, 8103-8116, 8183-8195, 8265-8278, 8349-8362,8425-8429, 9191-9376, 10307-10492, 11267-11413, 11765-11815,12071-12121, 12361-12407, 12479-12484, 12515-12520, 12551-12556,12587-12592, 12623-12628, or 12659-12661, or fragments or complementsthereof. In particular embodiments, the bisulfite-converted minus-strandcontrol DNA sequences include any one or more the sequences having atleast 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity to any of SEQ ID NOs: 8019-8032, 8103-8116, 8183-8195,12479-12484, 12515-12520, or 12551-12556, or fragments or complementsthereof. In some embodiments, the informative loci are associated withincreased methylation in both Barrett's esophagus and esophagealadenocarcinoma samples, as compared to the same sample types taken froma healthy control subject. In some embodiments, the disclosure providesfor bisulfite-treated sequences of any of the minus DNA strands that areassociated with increased methylation in both Barrett's esophagus andesophageal adenocarcinoma samples, as compared to the same sample typestaken from a healthy control subject. In some embodiments, the sequencesof any of the minus DNA strands that are associated with increasedmethylation in both Barrett's esophagus and esophageal adenocarcinomasamples are selected from the group consisting of sequences having atleast 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity to any of SEQ ID NOs: 1713-2140, 4281-4708, 6717-7111,7611-7636, 7767-7792, 7915-7938, 8019-8032, 8103-8116, 8183-8195,8265-8278, 8349-8362, or 8425-8429, or fragments or complements thereof.In particular embodiments, the sequences of any of the minus DNA strandsthat are associated with increased methylation in both Barrett'sesophagus and esophageal adenocarcinoma samples include any one or morebisulfite-converted methylated minus-strand DNA sequences selected fromthe group consisting of sequences having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to: SEQ IDNOs: 8019-8032, 8103-8116, 8183-8195, 8265-8278, 8349-8362, or8425-8429, or fragments or complements thereof. In some embodiments, thedisclosure provides for bisulfite-treated sequences of any of the minusDNA strands that are associated with increased methylation in anesophageal adenocarcinoma sample or a Barrett's with low grade or highgrade dysplasia sample as compared to a sample of the same type takenfrom a subject having Barret's esophagus without dysplasia. In someembodiments, the sequences of any of the minus DNA strands that areassociated with increased methylation in an esophageal adenocarcinomasample or a Barrett's with low grade or high grade dysplasia sampleinclude any one or more bisulfite-converted methylated minus-strand DNAsequences selected from the group consisting of sequences having atleast 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity to SEQ ID NOs. 9191-9376, 10307-10492, 11267-11413,11765-11815, 12071-12121, 12361-12407, 12479-12484, 12515-12520, or12551-12556, 12587-12592, 12623-12628, or 12659-12661, or fragments orcomplements thereof. In particular embodiments, the unmethylatedsequences of any of the minus DNA strands that are associated withincreased methylation in an esophageal adenocarcinoma sample or aBarrett's with low grade or high grade dysplasia sample include any oneor more of the sequences having at least 80%, 85%, 87%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs:12479-12484, 12515-12520, 12551-12556, 12587-12592, 12623-12628, or12659-12661, or fragments or complements thereof. In some embodiments,the informative loci are associated with reduced methylation in anesophageal adenocarcinoma sample as compared to a sample of the sametype taken from a subject having Barrett's esophagus. In someembodiments, the disclosure provides for methylated control sequences ofthe minus DNA strand that are associated with reduced methylation in anesophageal adenocarcinoma sample as compared to a sample of the sametype taken from a subject having Barrett's esophagus. In someembodiments, the methylated control sequences of any of the minus DNAstrands that are associated with reduced methylation in an esophagealadenocarcinoma sample include any one or more bisulfite-convertedmethylated minus-strand DNA sequences selected from the group consistingof sequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 13619-13855,15041-15277, 15607-15629, 15813-15852, 16053-16092, 16153-16157,16175-16177, 16192-16195 or 16203, or fragments or complements thereof.In particular embodiments, the methylated control sequences of any ofthe minus DNA strands that are associated with reduced methylation in anesophageal adenocarcinoma sample include any one or morebisulfite-converted methylated minus-strand DNA sequences selected fromthe group consisting of sequences having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ IDNOs: 16175-16177, 16192-16195 or 16203, or fragments or complementsthereof.

In some embodiments, the informative loci or amplicon of the informativeloci are treated with an agent, such as bisulfite. In some embodiments,the informative loci include sequences that have been treated withbisulfite, in some embodiments, the informative loci include methylatednucleic acid sequences that have been treated with bisulfite. In someembodiments, the bisulfite-converted methylated minus-strand DNAsequences include any one or more of sequences having at least 80%, 85%,87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identityto SEQ ID NOs: 2141-2568, 4709-5136, 7112-7506, 7637-7662, 7793-7818,7939-7962, 8033-8046, 8117-8130, 8196-8208, 8279-8292, 8363-8376,8430-8434, 9377-9562, 10493-10678, 11414-11560, 11816-11866,12122-12172, 12408-12454, 12485-12490, 12521-12526, 12557-12562,12593-12598, 12269-12634, or 12662-12664, or fragments or complementsthereof. In particular embodiments, the bisulfite-converted methylatedminus-strand DNA sequences include any one or more of sequences havingat least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100% identity to SEQ ID NOs: 8033-8046, 8117-8130, 8196-8208,8279-8292, 8363-8376, 8430-8434, 12485-12490, 12521-12526, 12557-12562,12593-12598, 12269-12634, or 12662-12664, or fragments or complementsthereof. In some embodiments, the informative loci are associated withincreased methylation in both Barrett's esophagus and esophagealadenocarcinoma samples, as compared to the same sample types taken froma healthy control subject. In some embodiments, the informative locithat are associated with increased methylation in both Barrett'sesophagus and esophageal adenocarcinoma samples include any one or morebisulfite-converted methylated minus-strand DNA sequences selected fromthe group consisting of sequences having at least 80%, 85%, 87%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ IDNOs: 2141-2568, 4709-5136, 7112-7506, 7637-7662, 7793-7818, 7939-7962,8033-8046, 8117-8130, 8196-8208, 8279-8292, 8363-8376, or 8430-8434, orfragments or complements thereof. In particular embodiments, theinformative loci that are associated with increased methylation in bothBarrett's esophagus and esophageal adenocarcinoma samples include anyone or more bisulfite-converted methylated minus-strand DNA sequencesselected from the group consisting of sequences having at least 80%,85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to: SEQ ID NOs: 8033-8046, 8117-8130, 8196-8208, 8279-8292,8363-8376, or 8430-8434, or fragments or complements thereof. In someembodiments, the informative loci are associated with increasedmethylation in an esophageal adenocarcinoma sample or a Barrett's withhigh grade dysplasia sample as compared to a sample of the same typetaken from a subject having Barrett's esophagus without dysplasia. Insome embodiments, the informative loci that are associated withincreased methylation in an esophageal adenocarcinoma sample or aBarrett's with low grade or high grade dysplasia sample include any oneor more bisulfite-converted methylated minus-strand DNA sequencesselected from the group consisting of sequences having at least 80%,85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to SEQ ID NOs: 9377-9562, 10493-10678, 11414-11560,11816-11866, 12122-12172, 12408-12454, 12485-12490, 12521-12526,12557-12562, 12593-12598, 12269-12634, or 12662-12664, or fragments orcomplements thereof. In particular embodiments, the informative locithat are associated with increased methylation in an esophagealadenocarcinoma sample or a Barrett's with low grade or high gradedysplasia sample include any one or more of the sequences of sequenceshaving at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identity to SEQ ID NOs: 12485-12490, 12521-12526,12557-12562, 12593-12598, 12269-12634, or 12662-12664, or fragments orcomplements thereof. In some embodiments, the informative loci areassociated with reduced methylation in an esophageal adenocarcinomasample as compared to a sample of the same type taken from a subjecthaving Barrett's esophagus. In some embodiments, the informative locithat are associated with reduced methylation in an esophagealadenocarcinoma sample include any one or more bisulfite-convertedmethylated minus-strand DNA sequences selected from the group consistingof sequences having at least 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs: 13856-14092,15278-15514, 15630-15652, 15853-15892, 16093-16132, 16158-16162,16178-16180, 16196-16198, or 16204, or fragments or complements thereof.In particular embodiments, the informative loci that are associated withreduced methylation in an esophageal adenocarcinoma sample include anyone or more of the sequences having at least 80%, 85%, 87%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NOs:16178-16180, 16196-16198, or 16204, or fragments or complements thereof.

In some embodiments, the disclosure provides for a bisulfite-convertednucleotide sequence comprising the bisulfite-converted nucleotidesequence of any one of the following-Up3, Up10, Up15-1, Up15-2, Up20-1,Up20-2, Up20-2, Up27, Up35-1, Up35-2, SqBE 2, SqBE5, SqBE7, SqBE9,SqBE10, SqBE11-1, SqBE11-2, SqBE13, SqBE14-2, SqBE 15, SqBE16-1,SqBE16-2, SqBE17-1, SqBE18, SqBE22-1, SqBE22-2 or SqBE23 In particularembodiments, the sequence comprises the bisulfite-converted nucleotidesequence of any one of the following: Up3, Up10, Up15-1, Up15-2, Up20-1,Up20-2, Up20-2, Up27, Up35-1, or Up35-2. In particular embodiments, thesequence comprises the bisulfite-converted nucleotide sequence of anyone of the following: SqBE2, SqBE5, SqBE7, SqBE9, SqBE10, SqBE11-1,SqBE11-2, SqBE13, SqBE14-2. SqBE15, SqBE16-1, SqBE16-2, SqBE17-1,SqBE18, SqBE22-1, SqBE22-2 or SqBE23.

In some embodiments, the disclosure provides for a panel of any of thesequences disclosed herein. In some embodiments, the panel comprises anyof the following combinations of sequences: a) Up3, Up10, Up15-1,Up15-2, Up20-1, Up20-2, Up27, Up35-1, and Up35-2; b) Up3, Up15-1,Up15-2, Up20-1, Up27, and Up35-1; c) Up10, Up3, Up15-1, Up15-2, Up20-1,Up27, and Up35-1; d) Up35-2, Up3, Up15-1, Up15-2, Up20-1, Up27 andUp35-1; e) Up15-1 and Up35-1; f) Up15-1, Up35-1, and Up10; g) Up15-1,Up35-1 and Up20-1; h) Up15-1, Up35-1, Up10, and Up15-2; i) Up15-1,Up35-1, Up10, and Up27; j) Up15-1, Up35-1, Up15-2, and Up20-1; k)Up15-1, Up35-1, Up15-2 and Up27:1) Up15-1, Up35-1, Up20-1, and Up27; m)Up3 and Up35-1; n) Up3 and Up35-2; o) Up3 and Up10; p) Up3 and Up27; q)Up35-1 and Up35-2; r) Up35-1 and Up27; s) Up35-2 and Up10; t) Up10 andUp27; u) Up3, Up35-1 and Up35-2; v) Up3, Up35-1 and Up10; w) Up3,Up35-1, and Up27; x) Up3, Up35-2 and Up10, y) Up3, Up35-2, and Up27; z)Up3, Up10, and Up27; aa) Up35-1, Up10, and Up27; ab) Up35-2, Up10, andUp27; ac) Up3, Up35-1, Up35-2 and Up10; ad) Up3, Up35-1, Up35-2 andUp27; ae) Up35-1, Up35-2, Up10 and Up27; af) Up3, Up35-2, Up10 and Up27;ag) Up3, Up35-1, Up10 and Up27; ah) Up3, Up10, Up27, Up35-1, and Up35-2;ai) Up35-1 and Up10, aj) Up35-1 and Up27; ak) Up35-2 and Up10; al)Up35-2 and Up27; am) Up3, Up35-1 and Up35-2; an) Up3, Up35-1, and Up10;ao) Up3, Up35-1, and Up27; ap) Up3, Up35-2 and Up10; aq) Up3, Up35-2 andUp27; ar) Up3, Up10 and Up27; at) Up35-1, Up10, and Up27; au) Up3,Up35-1, Up35-2, and Up10; av) Up3, Up35-1, Up35-2 and Up27; aw) Up35-1,Up35-2, Up10 and Up27; ax) Up3, Up35-2, Up10 and Up27; ay) Up3, Up35-1,Up10 and Up27; az) Up3, Up10, Up27, Up35-1, and Up35-2; ba) SqBE 5 andSqBE 7; bb) SqBE 5 and SqBE 16; be) SqBE 5 and SqBE 17; bd) SqBE 5 andSqBE18; be) SqBE 7 and SqBE 16; bf) SqBE 7 and SqBE 17; SqBE 7 and SqBE17; bg) SqBE 7 and SqBE18; bh) SqBE 16 and SqBE 17 and bi) SqBE 16 andSqBE18. In some embodiments, the disclosure provides for a method ofdetecting the methylation status of the sequences in any of the panelsdisclosed herein. In some embodiments, the disclosure provides for amethod of detecting the methylation status of the sequences in any ofthe panels disclosed herein, and further comprises detecting themutation status of p53. In particular embodiments, the disclosureprovides for a method of a) detecting the methylation status of a panelcomprising the sequences of Up-3 and Up35-2, and b) further detectingthe mutation status of TP53.

In some embodiments, the disclosure provides for a method of detectingthe methylation status of any of the loci disclosed herein, and furthercomprises detecting the methylation status of vimentin. In someembodiments, the vimentin methylation is detected in a manner consistentwith that described in Li et al. (Li M, et al. (2009) Sensitive digitalquantification of DNA methylation in clinical samples. Nat Biotechnol27(9):858-863). In some embodiments, the vimentin methylation patternsare determined in a nucleotide sequence having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ IDNO 16207 or 16208. In some embodiments, the methylation patterns aredetermined in any of the following nucleic acid sequence combinations:a) vimentin and SQBE5; b) vimentin and SQBE7, c) vimentin and SQBE16, d)vimentin and SQBE17 or e) vimentin and SQBE18.

In particular embodiments, the disclosure provides for a nucleotidesequence comprising a sequence having at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of thefollowing sequences: 8209-8222, 8251-8264, 8293-8306, 8335-8348,8405-8409, 8420-8424, 12563-12568, 12581-12586, 12599-12604,12617-12622, 12647-12649 or 12656-12658, or fragments and/or reversecomplements thereof. In particular embodiments, the disclosure providesfor a nucleotide sequence comprising a sequence having at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identityto any of the following sequences: 12563-12568, 12581-12586,12599-12604, 12617-12622, 12647-12649 or 12656-12658, or fragmentsand/or reverse complements thereof. In particular embodiments, thedisclosure provides for a nucleotide sequence comprising a sequencehaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity to any of the following sequences: 8209-8222,8251-8264, 8293-8306, 8335-8348, 8405-8409, or 8420-8424, or fragmentsand/or reverse complements thereof.

In some embodiments, the disclosure provides for a bisulfite-convertednucleotide sequence comprising a sequence having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any ofthe following sequences: SEQ ID NOs: 8307-8313, 8315-8327, 8329-8334,8349-8355, 8357-8369, 8371-8376, 8411, 8412, 8414, 8416, 8417, 8419,8426, 8427, 8429, 8431, 8432, 8434, 12605-12616, 12623-12634,12650-12655, or 12659-12664, or fragments and/or reverse complementsthereof. In some embodiments, the sequence comprises a sequence havingat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity to any of the following sequences: SEQ ID NOs: 8307-8313,8315-8327, 8329-8334, 8349-8355, 8357-8369, 8371-8376, 8411, 8412, 8414,8416, 8417, 8419, 8426, 8427, 8429, 8431, or 8432, 8434. In someembodiments, the sequence comprises a sequence having at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyof the following sequences: SEQ ID NOs: 12605-12616, 12623-12634,12650-12655, or 12659-12664.

In some embodiments, the disclosure provides for a bisulfite-convertednucleotide sequence comprising a sequence having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any ofthe following sequences. SEQ ID NOs: 8223-8250, 8265-8292, 12569-12580,or 12587-12598, or fragments and/or reverse complements thereof. In someembodiments, the sequence comprises a sequence having at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyof the following sequences: SEQ ID NOs: 8223-8250 or 8265-8292. In someembodiments, the sequence comprises a sequence having at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyof the following sequences: SEQ ID NOs: 12569-12580 or 12587-12598.

In some embodiments, the sample for use in any of the methods disclosedherein is a tissue sample taken from the subject. In some embodiments,the sample is tissue sample from the esophagus. In some embodiments, thesample is a biopsy or a brushing. In some embodiments, the sample is abiopsy or brushing of the esophagus. In some embodiments, the sample isa body fluid. In some embodiments, the body fluid is blood, serum,saliva, spit, stool, urine or an esophageal washing.

The present disclosure contemplates methods of selecting an individualto undergo a diagnostic procedure to determine the presence of Barrett'sesophagus, Barrett's esophagus with dysplasia (e.g., Barrett's esophaguswith low-grade or high-grade dysplasia), or of esophagealadenocarcinoma, by obtaining a biological sample from an individual, anddetermining in the sample the presence of DNA methylation in at leastone of any of the sequences disclosed herein. In some embodiments, thedisclosure provides for a method of selecting a subject for monitoringof esophageal neoplasia, wherein the presence of DNA methylation in atleast one of any of the sequences disclosed herein is detected in asample from the subject. In some embodiments, detection is achieved byany one or more of DNA sequencing, next generation sequencing,methylation specific PCR, methylation specific PCR combined with afluorogenic hybridization probe, real time methylation specific PCR, orhybridization to an array. In some embodiments, the detection in thesample is indicative that the subject is at high risk of progression toesophageal neoplasia (e.g., esophageal cancer) In some embodiments, thesubject is monitored by endoscopy. In some embodiments, a sample from asubject in which DNA methylation of at least one of any of the sequencesdisclosed herein is detected, is indicative that the subject should beadministered a particular treatment. In some embodiments, the treatmentis selected from the group consisting of endoscopic removal or ablationof an esophageal neoplasia, and/or surgery, radiation, or chemotherapytreatment of esophageal adenocarcinoma. In some embodiments, thesequence is any one or more sequence selected from the group consistingof a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identity to any of the following sequences:SEQ ID NOs: 1-856, 2569-3424, 5137-5926; 7507-7558, 7663-7714,7819-7866, 7963-7990, 8047-8074, 8131-8156, 8209-8222, 8251-8264,8293-8306, 8335-8348, 8405-8409, and 8420-8424, or fragments orcomplements thereof.

The present disclosure also contemplates methods of selecting anindividual to undergo a diagnostic procedure to determine presence ofBarrett's esophagus with low-grade dysplasia, Barrett's esophagus withhigh grade dysplasia or of esophageal adenocarcinoma, by obtaining abiological sample from an individual, and determining in the sample thepresence of DNA methylation in at least one of any of the sequencesdisclosed herein. In some embodiments, the sequence is any one or moresequence selected from the group consisting of sequences having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to any of the following sequences: SEQ ID NOs: 8447-8818,9563-9934, 10679-10972, 11561-11662, 11867-11968, 12173-12266,12455-12466, 12491-12502, 12527-12538, 12563-12568, 12581-12586,12599-12604, 12617-12622, 12647-12649, or 12656-12658, or fragments orcomplements thereof. The present disclosure further contemplates thatthe method may further comprise determining the status of somaticmutation(s) in TP53 in the sample. For example, a method comprisingdetermining the status of methylation of chromosomal loci e.g., Up15-1,Up35-1, Up35-2, Up3, Up27, and Up10 in a sample; and determining thepresence or absence of somatic mutation(s) in TP53 in the sample iscontemplated.

The present disclosure further contemplates that the method may furthercomprise determining the status of somatic mutation(s) in TP53 in thesample. For example, a method comprising determining the status ofmethylation of chromosomal loci e.g., Up15-1, Up35-1, Up35-2, Up3, Up27,and Up10 in a sample; and determining the presence or absence of somaticmutation(s) in TP53 in the sample is contemplated. In certainembodiments, the method optionally further comprises detecting thepresence or absence of a somatic mutation in TP53. In certainembodiments, the TP53 somatic mutation, as described herein, is any oneor more mutation at any one or more amino acid residue corresponding toamino acid residue 72, 105, 108, 110, 113, 124, 127, 132, 144, 152, 163,175, 183, 194, 213, 214, 218, 232, 234, 248, 265, 273, 278, 306, 337,347, or 639 of SEQ ID NO: 16205. In certain embodiments, the TP53somatic mutation is any non-synonymous somatic mutation known in theart. In certain embodiments, the TP53 somatic mutation is any one ormore mutation selected from the group consisting of Leu194Arg,Gly105Asp, Arg273His, Tyr163His, Ile232Thr, Arg213Ter, Arg273His,Arg248Gln, Arg175His, Arg110delinsGlnSer, Ser183Ter, Arg248Gln,Arg337Leu, Lys132Arg, Leu265ThrfsTer7, Arg306Ter, Cys124TrpfsTer25,Pro72Arg, Val218Glu, His214Leu, Gln144Ter, Phe113Scr, Tyr234His,Scr127Phe, Pro278Ala, Ala347Thr, and Pro152Leu of SEQ ID NO: 16205 Incertain embodiments, the TP53 mutation is any one or more non-synonymoussomatic mutation at any one or more nucleotide position corresponding tonucleotide position 108, 215, 314, 338, 380, 395, 430, 455, 487, 524,548, 581, 637, 639, 641, 653, 695, 700, 743, 818, 832, 916, 1010, or1039 of SEQ ID NO: 16206.

The present disclosure also contemplates methods of selecting anindividual to undergo a treatment for Barrett's esophagus, Barrett'sesophagus with low grade dysplasia. Barrett's esophagus with high gradedysplasia or for esophageal adenocarcinoma, by obtaining a biologicalsample from an individual, and determining in the sample the presence ofDNA methylation in at least one of any of the sequences disclosedherein. In some embodiments, the sequence is any one or more sequenceselected from the group consisting of sequence having at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyof the following sequences: SEQ ID NOs: 1-856, 2569-3424, 5137-5926;7507-7558, 7663-7714, 7819-7866, 7963-7990, 8047-8074, 8131-8156,8209-8222, 8251-8264, 8293-8306, 8335-8348, 8405-8409, or 8420-8424, orfragments or complements thereof. In some embodiments, the presentdisclosure further contemplates that the method may further comprisedetermining the status of somatic mutation(s) in TP53 in the sample. Forexample, a method comprising determining the status of methylation ofchromosomal loci e.g., Up15-1, Up35-1, Up35-2, Up3, Up27, and Up10 in asample; and determining the presence or absence of somatic mutation(s)in TP53 in the sample is contemplated.

The present disclosure also contemplates methods of selecting anindividual to undergo a treatment for Barrett's esophagus, Barrett'sesophagus with low-grade dysplasia, Barrett's esophagus with high gradedysplasia or for esophageal adenocarcinoma, by obtaining a biologicalsample from an individual, and determining in the sample the presence ofDNA methylation in at least one of any of the sequences disclosedherein. In some embodiments, the sequence is any one or more sequenceselected from the group consisting of sequence having at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to anyof the following sequences: SEQ ID NOs: 8447-8818, 9563-9934,10679-10972, 11561-11662, 11867-11968, 12173-12266, 12455-12466,12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604,12617-12622, 12647-12649, or 12656-12658, or fragments or complementsthereof. The present disclosure further contemplates that the method mayfurther comprise determining the status of somatic mutation(s) in TP53in the sample. For example, a method comprising determining the statusof methylation of chromosomal loci e.g, Up15-1, Up35-1, Up35-2, Up3,Up27, and Up10 in a sample; and determining the presence or absence ofsomatic mutation(s) in TP53 in the sample is contemplated. The presentdisclosure also contemplates methods of selecting an individual toundergo enhanced surveillance for the development of Barrett's esophaguswith low grade dysplasia. Barrett's esophagus with high grade dysplasiaor of esophageal adenocarcinoma, by obtaining a biological sample froman individual, and determining in the sample the presence of DNAmethylation in at least one of any of the sequences disclosed herein. Insome embodiments, the sequence is any one or more sequence selected fromthe group consisting of a sequence having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of thefollowing sequences: SEQ ID NOs: 8447-8818, 9563-9934, 10679-10972,11561-11662, 11867-11968, 12173-12266, 12455-12466, 12491-12502,12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622,12647-12649, or 12656-12658, or fragments or complements thereof. Thepresent disclosure further contemplates that the method may furthercomprise determining the status of somatic mutation(s) in TP53 in thesample. For example, a method comprising determining the status ofmethylation of chromosomal loci e.g., Up15-1, Up35-1, Up35-2, Up3, Up27,and Up10 in a sample; and determining the presence or absence of somaticmutation(s) in TP53 in the sample is contemplated. The presentdisclosure also contemplates methods of determining the response of anindividual with esophageal cancer to therapy by obtaining a biologicalsample from an individual with esophageal cancer, and determining thepresence of methylation in at least one of any of the sequencesdisclosed herein. In some embodiments, the sequence is any one or moresequence selected from the group consisting of sequences having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity to any of the following sequences: SEQ ID NOs: 1-856,2569-3424, 5137-5926; 7507-7558, 7663-7714, 7819-7866, 7963-7990,8047-8074, 8131-8156, 8209-8222, 8251-8264, 8293-8306, 8335-8348,8405-8409, or 8420-8424, 8447-8818, 9563-9934, 10679-10972; SEQ ID NOs:11561-11662, 11867-11968, 12173-12266; SEQ ID NOs: 12455-12466,12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604,12617-12622, 12647-12649, and 12656-12658, or fragments or complementsthereof. In some implementations, an increase in levels of methylationovertime is indicative of disease progression and a need for a change intherapy (such as modifying the dosing regime of an exiting therapy, oradministering a new therapeutic(s) either alone or in combination withthe existing therapy), and an absence of increase in levels ofmethylation over time or decrease in levels of methylation over time isindicative that a change in therapy is not required. The presentdisclosure further contemplates that the method may further comprisedetermining the status of somatic mutation(s) in TP53 in the sample. Forexample, a method comprising determining the status of methylation ofchromosomal loci e.g., Up15-1, Up35-1, Up35-2, Up3, Up27, and Up10 in asample; and determining the presence or absence of somatic mutation(s)in TP53 in the sample is contemplated.

The present disclosure also contemplates method of distinguishing EACand/or low/high grade dysplasia from BE by obtaining a biological samplefrom an individual, and determining in the sample the presence of DNAmethylation m at least one of any of the sequences disclosed herein. Insome embodiments, the sequence is any one or more sequence selected fromthe group consisting of sequences having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 6%, 970, 98%, 99% or 100% identity to any of thefollowing sequences: SEQ ID NOs: 8447-8818, 9563-9934, 10679-10972; SEQID NOs: 11561-11662., 11867-11968, 12173-12266; SEQ ID NOs: 12455-12466,12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604,12617-12622, 12647-12649, and 12656-12658, or fragments or complementsthereof. The present disclosure further contemplates that the method mayfurther comprise determining the status of somatic mutation(s) in TP53in the sample. For example, a method comprising determining the statusof methylation of chromosomal loci e.g., Up15-1, Up35-1, Up35-2, Up3,Up27, and Up10 in a sample; and determining the presence or absence ofsomatic mutation(s) in TP53 in the sample is contemplated. In certainembodiments, the absence of methylation at Up15-1, Up35-1, Up35-2, Up3,Up27, and Up10; and the absence of a somatic mutation in TP53 may beindicative of non-dysplastic Barret's esophagus. In certain embodiments,the presence of methylation at any one of Up15-1, Up35-1, Up35-2, Up3,Up27, and Up10; or the presence of a somatic mutation in TP53 may beindicative of esophageal adenocarcinoma or of Barrett's with high gradedysplasia.

The present disclosure also contemplates method of distinguishing EACand/or low/high grade dysplasia from BE by obtaining a biological samplefrom an individual, and determining in the sample the presence of DNAmethylation in at least one of any of the sequences disclosed herein. Insome embodiments, the sequence is any one or more sequence selected fromthe group consisting of sequences having at least 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of thefollowing sequences: SEQ ID NOs: 8447-8818, 9563-9934, 10679-10972, SEQID NOs: 11561-11662, 11867-11968, 12173-12266; SEQ ID NOs: 12455-12466,12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604,12617-12622, 12647-12649, and 12656-12658, or fragments or complementsthereof. The present disclosure further contemplates that the method mayfurther comprise determining the status of somatic mutation(s) in TP53in the sample. For example, a method comprising determining the statusof methylation of chromosomal loci e.g., Up15-1, Up35-1, Up35-2, Up3,Up27, and Up10 in a sample; and determining the presence or absence ofsomatic mutation(s) in TP53 in the sample is contemplated. In certainembodiments, the absence of methylation at Up15-1, Up35-1, Up35-2, Up3,Up27, and Up10; and the absence of a somatic mutation in TP53 may beindicative of non-dysplastic Barret's esophagus. In certain embodiments,the presence of methylation at any one of Up15-1, Up35-1, Up35-2, Up3,Up27, and Up10; or the presence of a somatic mutation in TP53 may beindicative of esophageal adenocarcinoma.

The present disclosure also contemplates a method of monitoring theprogression (or regression) of esophageal neoplasias over time. Themethod involves detecting the methylation status of one or morenucleotide sequences selected from the group consisting of sequenceshaving at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity to any of the following sequences: SEQ ID NOs:1-856, 2569-3424, 5137-5926; 7507-7558, 7663-7714, 7819-7866, 7963-7990,8047-8074, 8131-8156, 8209-8222, 8251-8264, 8293-8306, 8335-8348,8405-8409, and 8420-8424, 8447-8818, 9563-9934, 10679-10972; SEQ ID NOs:11561-11662, 11867-11968, 12173-12266; SEQ ID NOs: 12455-12466,12491-12502, 12527-12538, 12563-12568, 12581-12586, 12599-12604,12617-12622, 12647-12649, and 12656-12658, or fragments or complementsthereof in samples from a subject at a first time and at a later time.In certain embodiments, neoplasia regression may be indicated by theabsence of methylation in the nucleotide sequence taken at a later timeand the presence of methylation in the nucleotide sequence taken at thefirst time. In certain embodiments, neoplasia progression may beindicated by the presence of methylation in the nucleotide sequencetaken at a later time and the absence of methylation in the nucleotidesequence taken at the first time. The present disclosure furthercontemplates that the method may further comprise determining the statusof somatic mutation(s) in TP53 in the samples. In some embodiments,neoplastic regression may be indicated by the presence of methylatedchromosomal loci e.g., methylation of Up15-1, Up35-1, Up35-2, Up3, Up27,and/or Up10 or the presence of a somatic mutation in TP53 in a firstsample; and the absence of methylated chromosomal loci e.g.,unmethylated Up15-1, Up35-1, Up35-2, Up3, Up27, and Up10, and theabsence of somatic mutation(s) in TP53 in a later sample. In someembodiments, neoplastic progression may be indicated by the presence ofunmethylated chromosomal loci e.g., unmethylated Up15-1, Up35-1, Up35-2,Up3, Up27, and Up10; and the absence of somatic mutation(s) in TP53 in afirst sample; and the presence of methylated chromosomal loci e.g.,methylated Up15-1, Up35-1, Up35-2, Up3, Up27, and/or Up10, or thepresence of a somatic mutation in TP53 in a later sample.

The present disclosure also provides sequences that will hybridize underhighly stringent conditions to the nucleotide sequences of any one ormore of SEQ ID NOs: 1-8444 and 8447-16204, or fragments or complementsthereof. As discussed above, one of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied One of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C., to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

In other embodiments, the disclosure also provides the methylated formsof the nucleotide sequences of any one or more of SEQ ID NOs: 1-8444 and8447-16204, or fragments thereof, wherein the cytosine bases of the CpGislands present in the sequences are methylated. In other words, thenucleotide sequences listed of any one or more of SEQ ID NOs: 1-8444 or8447-16204 or fragments or complements thereof may be either in themethylated status (e.g., as seen in neoplasias) or in the unmethylatedstatus (e.g., as seen in normal cells). In further embodiments, thenucleotide sequences of the disclosure can be isolated, recombinant,and/or fused with a heterologous nucleotide sequence, or in a DNAlibrary.

In certain embodiments, the present disclosure providesbisulfite-converted nucleotide sequences, for example,bisulfite-converted sequences selected from any of the sequencesdisclosed herein. In some embodiments, the sequence is selected from thegroup consisting of sequences having at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of thefollowing sequences: SEQ ID NOs: 857-2568, 3425-5136, 5927-7506,7559-7662, 7715-7818, 7867-7962, 7991-8046, 8075-8130, 8157-8208,8223-8250, 8265-8292, 8307-8334, 8349-8376, 8410-8419, 8425-8434, and/orfragments thereof, and/or the reverse complements thereof. In yet otherembodiments, the disclosure provides bisulfite-converted sequencesselected from the group consisting of sequences having at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identityto any of the following sequences: SEQ ID NOs: 8819-9562, 9935-10678,10973-11560, 11663-11866, 11969-12172, 12267-12454; 12467-12490,12503-12526, 12539-12562, 12569-12580, 12587-12598, 12605-12616,12623-12634, 12650-12655, and 12659-12664, and/or fragments thereof,and/or the reverse complements thereof. In yet other embodiments, thedisclosure provides bisulfate-converted sequences selected from thegroup consisting of sequences having at least 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any of thefollowing sequences: SEQ ID NOs: 13145-14092, 14567-15514, 15561-15652;15733-15892, 15973-16132, 16143-16162; 16169-16180, 16187-16198, and16201-16204, and/or fragments thereof, and/or the reverse complementsthereof.

A fragment/portion of any of the nucleotide sequences disclosed hereinmay be of any length, so long as the methylation status of thatnucleotide sequence may be determined. In some embodiments, thenucleotide sequence is at least 10, 15, 25, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1500, 1700, or 2000nucleotides in length, in some embodiments, the nucleotide sequence isat least 10-2000, 10-1000, 10-500, 10-200, 10-150, 10-100, 50-2000,50-1000, 50-500, 50-200, 50-150, 50-100, 80-2000, 80-1000, 80-500,80-150, 80-100, 100-2000, 100-1000, 100-500, 100-200, or 100-150nucleotides in length.

Such bisulfite-converted nucleotide sequences can be used for detectingthe methylation status, for example, by an MSP reaction or by directsequencing (e.g., next generation sequencing). These bisulfite-convertedsequences are also of use for designing primers for MSP reactions thatspecifically detect methylated or uunethylated nucleotide sequencesfollowing bisulfite conversion. In yet other embodiments, thebisulfite-converted nucleotide sequences of the disclosure also includenucleotide sequences that will hybridize under highly stringentconditions to any nucleotide sequence of any one or more of SEQ ID NOs:1-8444 and 8447-16204, or fragments or complements thereof.

In further aspects, the application provides methods for producing suchbisulfite-converted nucleotide sequences, for example, the applicationprovides methods for treating a nucleotide sequence with a bisulfiteagent such that the unmethylated cytosine bases are converted to adifferent nucleotide base such as a uracil.

In yet other aspects, the application provides oligonucleotide primersfor amplifying a region within the nucleic acid sequence of any one ormore of SEQ ID NOs: 1-8444 and 8447-16204. In certain aspects, a pair ofthe oligonucleotide primers can be used in a detection assay, such asthe HpaII assay. In certain aspects, primers used in an MSP reaction canspecifically distinguish between methylated and non-methylated DNA.

The primers of the disclosure have sufficient length and appropriatesequence so as to provide specific initiation of amplification nucleicacids. Primers of the disclosure are designed to be “substantially”complementary to each strand of the nucleic acid sequence to beamplified. In some embodiments, the primer is selected from the groupconsisting of sequences having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identity to any of SEQ ID NOs: 8377-8404,8435-8446, 12635-12646, and 12665-12670. In some embodiments, the primercomprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, or 35 consecutive nucleotides of any of the primer sequences of SEQID NOs: 8377-8404, 8435-8446, 12635-12646, and 12665-12670. Whileexemplary primers include the sequences of any sequence having at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity to any of SEQ ID NOs: 8377-8404, 8435-8446, 12635-12646, and12665-12670, or fragments thereof, it is understood that any primersthat hybridize with the bisulfite-converted sequence of any one or moreof SEQ ID NOs: 1-8444 and 8447-16204 are included within the scope ofthis disclosure and is useful in the method of the disclosure fordetecting methylated nucleic acid, as described. Similarly, it isunderstood that any primers that would serve to amplify a methylationsensitive restriction site or sites within the differentially methylatedregion of the informative loci of any of the sequences of SEQ ID NOs:1-8444 or 8447-16204, or fragments or complements thereof are includedwithin the scope of this disclosure and is useful in the method of thedisclosure for detecting nucleic methylated nucleic acid, as described.

The oligonucleotide primers of the disclosure may be prepared by usingany suitable method, such as conventional phosphotriester andphosphodiester methods or automated embodiments thereof. In one suchautomated embodiment, diethylphosphoramidites are used as startingmaterials and may be synthesized as described by Beaucage, et al.(Tetrahedron Letters, 22:1859-1862, 1981). One method of synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066.

IV. Assays and Drug Screening Methodologies

In certain aspects, the application provides assays and methods usingany of the informative loci, or bisulfite converted methylated orunmethylated sequences thereof, disclosed herein, in some embodiments,the informative loci comprise a sequence having at least 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any ofthe sequences of SEQ ID NOs: 1-428, 2569-2996, 5137-5531, 7507-7532,7663-7668, 7819-7842, 7963-7976, 8047-8060, 8131-8143, 8209-8222,8293-8306, 8405-8409, 8447-8632, 9563-9748.10679-10825, 11561-11611,11867-11917, 12173-12219, 12455-12460, 12491-12496, 12527-12532,12563-12568, 12599-12604, 12647-12649, 12671-12908, 14093-14329,15515-15537, 15653-15692, 15893-15932, 16133-16137, 16163-16165,16181-16183, 16199, 429-856, 2997-3424, 5532-5926, 7533-7558, 7689-7714,7843-7866, 7977-7990, 8061-8074, 8144-8156, 8251-8264, 8335-8348,8420-8424, 8633-8818, 9749-9934, 10826-10972, 11612-11662, 11918-11968,12220-12266, 12461-12466, 12497-12502, 12533-12538, 12581-12586,12617-12622, 12656-12658, 12909-13144, 14330-14566, 15538-15560,15693-15732, 15933-15972, 16138-16142, 16166-16168, 16184-16186 or 16200or any fragments or complements thereof. In some embodiments, theinformative loci are used as molecular markers to distinguish betweenhealthy cells and metaplastic cells (e.g, Barrett's esophageal cells) Insome embodiments, the informative loci are used as molecular markers todistinguish between healthy cells and neoplastic cells (e.g., cancercells). In particular embodiments, the informative loci are used asmolecular markers to distinguish between healthy cells and esophagealadenocarcinoma cells. In some embodiments, the informative loci are usedas molecular markers to distinguish between Barrett's esophagus cellsand cancer cells In some embodiments, the informative loci are used asmolecular markers to distinguish between Barrett's esophagus cells andesophageal adenocarcinoma cells. For example, in one embodiment, theapplication provides methods and assays using any of the informativeloci comprising a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQID NOs: 8447-8818, 9563-9934, 10679-10972, 11561-11662, 11867-11968,12173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568,12581-12586, 12599-12604, 12617-12622, 12647-12649, OR 12656-12658, orany fragments or complements thereof as markers that distinguish betweenhealthy cells and neoplasia cells. In other embodiments, the applicationprovides methods and assays using the informative loci comprising asequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identity to any one or more of SEQ ID NOs: 1-856,2569-3424, 5137-5926, 7507-7558, 7663-7714, 7819-7866, 7963-7990,8047-8047, 8131-8156, 8209-8222, 8251-8264, 8293-8306, 8335-8348,8405-8409, 8420-8424, 8447-8818, 9563-9934, 10679-10972, 11561-11662,11867-11968, 12173-12266, 12455-12466, 12491-12502, 12527-12538,12563-12568, 12581-12586, 12599-12604, 12617-12622, 12647-12649,12656-12658, 12671-13144, 14093-14566, 15515-15560, 15653-15732,15893-15972, 16135-16142, 16163-16168, 16181-16186 and/or 16199-16200 orany fragments or complements thereof as markers that distinguish betweenhealthy cells and cells derived from neoplasias of the uppergastrointestinal tract. In one aspect, a molecular marker of theinvention is a differentially, methylated sequence of an informativelocus. In certain aspects, the application provides assays and methodsusing the informative loci comprising a sequence having at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identityto any one or more of SEQ ID NOs: 1-856, 2569-3424, 5137-5926,7507-7558, 7663-7714, 7819-7866, 7963-7990, 8047-8047, 8131-8156,8209-8222, 8251-8264, 8293-8306, 8335-8348, 8405-8409, 8420-8424,8447-8818, 9563-9934, 10679-10972, 11561-11662, 11867-11968,12173-12266, 12455-12466, 12491-12502, 12527-12538, 12563-12568,12581-12586, 12599-12604, 12617-12622, 12647-12649, 12656-12658,12671-13144, 14093-14566, 15515-15560, 15653-15732, 15893-15972,16135-16142, 16163-16168, 16181-16186 and/or 16199-16200 or anyfragments or complements thereof in combination with the status ofsomatic mutation(s) in TP53 as molecular markers that distinguishbetween healthy cells and cancer cells. For example, in one embodiment,the application provides methods and assays using the informative locicomprising a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity to any one or more of SEQ IDNOs: 1-856, 2569-3424, 5137-5926, 7507-7558, 7663-7714, 7819-7866,7963-7990, 8047-8047, 8131-8156, 8209-8222, 8251-8264, 8293-8306,8335-8348, 8405-8409, 8420-8424, 8447-8818, 9563-9934, 10679-10972,11561-11662, 11867-11968, 12173-12266, 12455-12466, 12491-12502,12527-12538, 12563-12568, 12581-12586, 12599-12604, 12617-12622,12647-12649, 12656-12658, 12671-13144, 14093-14566, 15515-15560,15653-15732, 15893-15972, 16135-16142, 16163-16168, 16181-16186 and/or16199-16200 or any fragments or complements thereof and the status ofsomatic mutation(s) in TP53 as markers that distinguish between healthycells and neoplasia cells (e.g. cancer/esophageal adenocarcinoma cells).In other embodiments, the application provides methods and assays usingthe informative loci disclosed herein (e.g., chromosomal loci Up15-1,Up35-1, Up35-2, Up3, Up27, and Up10) and the status of somaticmutation(s) in TP53 as markers that distinguish between healthy cellsand cells derived from neoplasias of the upper gastrointestinal tract.In one aspect, a molecular marker of the invention is a differentiallymethylated sequence of an informative locus.

In certain embodiments, the application provides assays for detectingdifferentially methylated nucleotide sequences (e.g., vimentin and/orSqBE18). Thus, a differentially methylated nucleotide sequence, in itsmethylated state, can serve as a target for detection using variousmethods described herein and the methods that are well within thepurview of the skilled artisan in view of the teachings of thisapplication.

In certain aspects, such methods for detecting methylated nucleotidesequences (e.g., vimentin and/or SqBE18) are based on treatment ofgenomic DNA with a chemical compound which converts non-methylated C,but not methylated C (i.e., 5 mC), to a different nucleotide base. Onesuch compound is sodium bisulfite (also referred to simply as“bisulfite” herein), which converts C, but not 5 mC, to U. Methods forbisulfite treatment of DNA are known in the art (Herman, et al., 1996,Proc Natl Acad Sci USA, 93:9821-6; Herman and Baylin, 1998, CurrentProtocols in Human Genetics, N. E. A. Dracopoli, ed., John Wiley & Sons,2:10.6.1-10.6.10; U.S. Pat. No. 5,786,146). To illustrate, when a DNAmolecule that contains unmethylated C nucleotides is treated with sodiumbisulfite to become a compound-converted DNA, the sequence of that DNAis changed (C→U). Detection of the U in the converted nucleotidesequence is indicative of an unmethylated C.

The different nucleotide base (e.g., U) present in compound-convertednucleotide sequences can subsequently be detected in a variety of ways.In a particular embodiment, the present invention provides a method ofdetecting U in compound-converted DNA sequences by using “methylationsensitive PCR” (MSP) (see, e.g., Herman, et al., 1996, Proc. Natl. Acad.Sci. ISA, 93:9821-9826; U.S. Pat. Nos. 6,265,171; 6,017,704; 6,200,756).In MSP, one set of primers (i.e., comprising a forward and a reverseprimer) amplifies the compound-converted template sequence if C bases inCpG dinucleotides within the DNA are methylated. This set of primers iscalled “methylation-specific primers.” Another set of primers amplifiesthe compound-converted template sequence if C bases in CpG dinucleotideswithin the 5′ flanking sequence are not methylated. This set of primersis called “unmethylation-specific primers.”

In MSP, the reactions use the compound-converted DNA from a sample in asubject In assays for methylated DNA, methylation-specific primers areused. In the case where C within CpG dinucleotides of the targetsequence of the DNA are methylated, the methylation-specific primerswill amplify the compound-converted template sequence in the presence ofa polymerase and an MSP product will be produced. If C within CpGdinucleotides of the target sequence of the DNA is not methylated, themethylation-specific primers will not amplify the compound-convertedtemplate sequence in the presence of a polymerase and an MSP productwill not be produced. In some embodiments, any of the bisulfiteconverted methylated sequences disclosed herein is used as a marker fora particular indication.

It is often also useful to run a control reaction for the detection ofunmethylated DNA. The reaction uses the compound-converted DNA from asample in a subject and unmethylation-specific primers are used. In thecase where C within CpG dinucleotides of the target sequence of the DNAare unmethylated, the unmethylation specific primers will amplify thecompound-converted template sequence in the presence of a polymerase andan MSP product will be produced. If C within CpG dinucleotides of thetarget sequence of the DNA is methylated, the unmethylation-specificprimers will not amplify the compound-converted template sequence in thepresence of a polymerase and an MSP product will not be produced. Notethat a biologic sample will often contain a mixture of both neoplasticcells that give rise to a signal with methylation specific primers, andnormal cellular elements that give rise to a signal withunmethylation-specific primers. The unmethylation specific signal isoften of use as a control reaction, but does not in this instance implythe absence of neoplasia as indicated by the positive signal derivedfrom reactions using the methylation specific primers In someembodiments, any of the bisulfite converted unmethylated sequencesdisclosed herein are used as controls. In some embodiments, theunmethylated control sequences are any of the bisulfite convertedsequences of SEQ ID NOs: 857-1284, 3425-3852, 5927-6321, 7559-7584,7715-7740, 7867-7890, 7991-8004, 8075-8088, 8157-8169, 8223-8236,8307-8320, 8410-8414, 8819-9004, 9935-10120, 10973-11119, 11663-11713,11969-12019, 12267-12313, 12467-12472, 12503-12508, or 12539-12544,12569-12574, 12605-12610, 12650-12652, 1713-2140, 4281-4708, 6717-7111,7611-7636, 7767-7792, 7915-7938, 8019-8032, 8103-8116, 8183-8195,8265-8278, 8349-8362, 8425-8429, 9191-9376, 10307-10492, 11267-11413,11765-11815, 12071-12121, 12361-12407, 12479-12484, 12515-12520,12551-12556, 12587-12592, 12623-12628, or 12659-12661 in which every “Y”position is a “T.”

Primers for a MSP reaction are derived from the compound-convertedtemplate sequence. Herein, “derived from” means that the sequences ofthe primers are chosen such that the primers amplify thecompound-converted template sequence in a MSP reaction. Each primercomprises a single-stranded DNA fragment which is at least 8 nucleotidesin length. In some embodiments, the primers are less than 50 nucleotidesin length, or in some embodiments, from 15 to 35 nucleotides in length.Because the compound-converted template sequence can be either theWatson strand or the Crick strand of the double-stranded DNA that istreated with sodium bisulfite, the sequences of the primers is dependentupon whether the Watson or Crick compound-converted template sequence ischosen to be amplified in the MSP. Either the Watson or Crick strand canbe chosen to be amplified.

The compound-converted template sequence, and therefore the product ofthe MSP reaction, is, in some embodiments, between 20 to 3000nucleotides in length. In other embodiments, the product of the MSPreaction is between 50 to 1000 nucleotides in length. In otherembodiments, the product of the MSP reaction is between 50 to 500nucleotides in length in other embodiments, the product of the MSPreaction is between 80-150 nucleotides in length. In some embodiments,the product of the MSP reaction is at least 20, 30, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240 or 250 nucleotides in length. In some embodiments, themethylation-specific primers result in an MSP product of a differentlength than the MSP product produced by the unmethylation-specificprimers.

A variety of methods can be used to determine if an MSP product has beenproduced in a reaction assay. One way to determine if an MSP product hasbeen produced in the reaction is to analyze a portion of the reaction byagarose gel electrophoresis. For example, a horizontal agarose gel offrom 0.6 to 2.0% agarose is made and a portion of the MSP reactionmixture is electrophoresed through the agarose gel. Afterelectrophoresis, the agarose gel is stained with ethidium bromide MSPproducts are visible when the gel is viewed during illumination withultraviolet light. By comparison to standardized size markers, it isdetermined if the MSP product is of the correct expected size.

Other methods can be used to determine whether a product is made in anMSP reaction. One such method is called “real-time PCR.” Real-time PCRutilizes a thermal cycler (i.e., an instrument that provides thetemperature changes necessary for the PCR reaction to occur) thatincorporates a fluorimeter (i.e, an instrument that measuresfluorescence). The real-time PCR reaction mixture also contains areagent whose incorporation into a product can be quantified and whosequantification is indicative of copy number of that sequence in thetemplate. One such reagent is a fluorescent dye, called SYBR Green I(Molecular Probes, Inc.; Eugene, Oreg.) that preferentially bindsdouble-stranded DNA and whose fluorescence is greatly enhanced bybinding of double-stranded DNA. When a PCR reaction is performed in thepresence of SYBR Green I, resulting DNA products bind SYBR Green I andfluorescence. The fluorescence is detected and quantified by thefluorimeter. Such technique is particularly useful for quantification ofthe amount of the product in the PCR reaction. Additionally, the productfrom the PCR reaction may be quantitated in “real-time PCR” by the useof a variety of probes that hybridize to the product including TaqManprobes and molecular beacons. Quantitation may be on an absolute basis,or may be relative to a constitutively methylated DNA standard, or maybe relative to an unmethylated DNA standard. In one instance the ratioof methylated derived product to unmethylated derived product may beconstructed.

Methods for detecting methylation of the DNA according to the presentdisclosure are not limited to MSP, and may cover any assay for detectingDNA methylation. Another example method of detecting methylation of theDNA is by using “methylation-sensitive” restriction endonucleases. Suchmethods comprise treating the genomic DNA isolated from a subject with amethylation-sensitive restriction endonuclease and then using therestriction endonuclease-treated DNA as a template in a PCR reaction.Herein, methylation-sensitive restriction endonucleases recognize andcleave a specific sequence within the DNA if C bases within therecognition sequence are not methylated. If C bases within therecognition sequence of the restriction endonuclease are methylated, theDNA will not be cleaved. Examples of such methylation-sensitiverestriction endonucleases include, but are not limited to HpaII, SmaI,SacII, EagI, BstUI, and BssHII. In this technique, a recognitionsequence for a methylation-sensitive restriction endonuclease is locatedwithin the template DNA, at a position between the forward and reverseprimers used for the PCR reaction. In the case that a C base within themethylation-sensitive restriction endonuclease recognition sequence isnot methylated, the endonuclease will cleave the DNA template and a PCRproduct will not be formed when the DNA is used as a template in the PCRreaction. In the case that a C base within the methylation-sensitiverestriction endonuclease recognition sequence is methylated, theendonuclease will not cleave the DNA template and a PCR product will beformed when the DNA is used as a template in the PCR reaction.Therefore, methylation of C bases can be determined by the absence orpresence of a PCR product (Kane, et al., 1997, Cancer Res, 57:808-11).No sodium bisulfite is used in this technique.

Yet another exemplary method of detecting methylation of the DNA iscalled the modified MSP, which method utilizes primers that are designedand chosen such that products of the MSP reaction are susceptible todigestion by restriction endonucleases, depending upon whether thecompound-converted template sequence contains CpG dinucleotides or UpGdinucleotides.

Yet other methods for detecting methylation of the DNA include theMS-SnuPE methods. This method uses compound-converted DNA as a templatein a primer extension reaction wherein the primers used produce aproduct, dependent upon whether the compound-converted template containsCpG dinucleotides or UpG dinucleotides (see e.g., Gonzalgo, et al.,1997, Nucleic Acids Res., 25:2529-31).

Another exemplary method of detecting methylation of the DNA is calledCOBRA (i.e., combined bisulfite restriction analysis). This method hasbeen routinely used for DNA methylation detection and is well known inthe art (see, e.g., Xiong, et al., 1997, Nucleic Acids Res, 25:25324).In this technique, methylation-sensitive restriction endonucleasesrecognize and cleave a specific sequence within the DNA if C baseswithin the recognition sequence are methylated. If C bases within therecognition sequence of the restriction endonuclease are not methylated,the DNA will not be cleaved. In some embodiments, the method utilizesmethylation-sensitive restriction endonucleases.

Another exemplary method of detecting methylation of DNA requireshybridization of a compound converted DNA to arrays that include probesthat hybridize to sequences derived from a methylated template.

Another exemplary method of detecting methylation of DNA includesprecipitation of methylated DNA with antibodies that bind methylated DNAor with other proteins that bind methylated DNA, and then detection ofDNA sequences in the precipitate. The detection of DNA could be done byPCR based methods, by hybridization to arrays, or by other methods knownto those skilled in the art.

Another exemplary method of detecting methylated DNA is bisulfitesequencing that involves amplification of a target region of bisulfiteconverted DNA using methylation indifferent PCR primers that amplifyconverted DNAs derived from both methylated and unmethylated templates.The methylation indifferent primers are often designed to be bothmethylation indifferent and bisulfite specific, i.e, to amplify onlybisulfite converted target DNAs and not to amplify non-converted targetsequences. In some embodiments, the amplified DNAs then may becharacterized by Next Generation Sequencing methods that allow eachcytosine base in the original template to be assessed within each DNAsequence read for the presence of methylation (retention of cytosine) orthe absence of methylation (conversion to thymidine). The percent ofmethylation at each cytosine base in the original template can then becalculated by the percent of DNA reads in which the cytosine ispreserved as cytosine versus is converted to thymidine. Similarly, thepercent of methylation across a region of interest can be assessed bydetermining a rule for assessing the region as methylated orunmethylated in an individual DNA read (i.e. determining a cutoff formethylation in the region that will categorize the region as“methylated”), and then determining the percent of DNA reads in whichthe region qualifies as methylated.

In certain embodiments, the disclosure provides methods that involvedirectly sequencing the product resulting from an MSP reaction todetermine if the compound-converted template sequence contains CpGdinucleotides or UpG dinucleotides. Molecular biology techniques such asdirectly sequencing a PCR product are well known in the art In someembodiments, methylation of DNA may be measured as a percentage of totalDNA. High levels of methylation may be 1-100% methylation, for example,1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% methylation Lowlevels of methylation may be 0%-0.99% methylation, for example, 0%,0.1%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%. At least some normal tissues, forexample, normal esophagus samples, may not have any detectablemethylation.

The skilled artisan will appreciate that the present disclosure is basedin part, on the recognition that any one of the informative locidisclosed herein may include nucleotide sequences that encodepolypeptides that, for example, may function as a tumor suppressor gene.Accordingly, the application further provides methods for detecting suchpolypeptides in cell samples. In some embodiments, the disclosureprovides detection methods by assaying such polypeptides so as todetermine whether a patient has or does not have a disease condition.Further, such a disease condition may be characterized by decreasedlevels of such polypeptides. In certain embodiments, the disclosureprovides methods for determining whether a patient is or is not likelyto have cancer by detecting such polypeptides. In further embodiments,the disclosure provides methods for determining whether the patient ishaving a relapse or determining whether a patient's cancer is respondingto treatment.

Optionally, such methods involve obtaining a quantitative measure of theprotein in the sample. In view of this specification, one of skill inthe art will recognize a wide range of techniques that may be employedto detect and optionally quantitate the presence of a protein. In someembodiments, a protein is detected with an antibody. In manyembodiments, an antibody-based detection assay involves bringing thesample and the antibody into contact so that the antibody has anopportunity to bind to proteins having the corresponding epitope. Inmany embodiments, an antibody-based detection assay also typicallyinvolves a system for detecting the presence of antibody-epitopecomplexes, thereby achieving a detection of the presence of the proteinshaving the corresponding epitope. Antibodies may be used in a variety ofdetection techniques, including enzyme-linked immunosorbent assays(ELISAs), immunoprecipitations, Western blots. Antibody-independenttechniques for identifying a protein may also be employed. For example,mass spectroscopy, particularly coupled with liquid chromatography,permits detection and quantification of large numbers of proteins in asample. Two-dimensional gel electrophoresis may also be used to identifyproteins, and may be coupled with mass spectroscopy or other detectiontechniques, such as N-terminal protein sequencing. RNA aptamers withspecific binding for the protein of interest may also be generated andused as a detection reagent. Samples should generally be prepared in amanner that is consistent with the detection system to be employed. Forexample, a sample to be used in a protein detection system shouldgenerally be prepared in the absence of proteases. Likewise, a sample tobe used in a nucleic acid detection system should generally be preparedin the absence of nucleases. In many instances, a sample for use in anantibody-based detection system will not be subjected to substantialpreparatory steps. For example, urine may be used directly, as maysaliva and blood, although blood will, in certain embodiments, beseparated into fractions such as plasma and serum.

In certain embodiments, a method of the disclosure comprises detectingthe presence of an informative loci-expressed nucleic acid, such as anmRNA, in a sample. Optionally, the method involves obtaining aquantitative measure of the informative loci-expressed nucleic acid inthe sample. In view of this specification, one of skill in the art willrecognize a wide range of techniques that may be employed to detect andoptionally quantitate the presence of a nucleic acid. Nucleic aciddetection systems generally involve preparing a purified nucleic acidfraction of a sample, and subjecting the sample to a direct detectionassay or an amplification process followed by a detection assay.Amplification may be achieved, for example, by polymerase chain reaction(PCR), reverse transcriptase (RT) and coupled RT-PCR. Detection of anucleic acid is generally accomplished by probing the purified nucleicacid fraction with a probe that hybridizes to the nucleic acid ofinterest, and in many instances, detection involves an amplification aswell. Northern blots, dot blots, microarrays, quantitative PCR, andquantitative RT-PCR are all well known methods for detecting a nucleicacid in a sample.

In certain embodiments, the disclosure provides nucleic acid probes thatbind specifically to an informative loci nucleic acid. Such probes maybe labeled with, for example, a fluorescent moiety, a radionuclide, anenzyme or an affinity tag such as a biotin moiety. For example, theTaqMan® system employs nucleic acid probes that are labeled in such away that the fluorescent signal is quenched when the probe is free insolution and bright when the probe is incorporated into a larger nucleicacid.

Immunoscintigraphy using monoclonal antibodies directed at theinformative loci may be used to detect and/or diagnose a cancer. Forexample, monoclonal antibodies against the informative loci labeled with⁹⁹Technetium, ¹¹¹Indium, ¹²⁵Iodine-may be effectively used for suchimaging. As will be evident to the skilled artisan, the amount ofradioisotope to be administered is dependent upon the radioisotope.Those having ordinary skill in the art can readily formulate the amountof the imaging agent to be administered based upon the specific activityand energy of a given radionuclide used as the active moiety. Typically0.1-100 millicuries per dose of imaging agent, 1-10 millicuries, oroften 2-5 millicuries are administered. Thus, compositions according tothe present invention useful as imaging agents comprising a targetingmoiety conjugated to a radioactive moiety comprise 0.1-100 millicuries,in some embodiments 1-10 millicuries, in some embodiments 2-5millicuries, in some embodiments 1-5 millicuries.

A variety of methods can be used to determine if TP53 contains a somaticmutation, as will be evident to the skilled artisan. In someembodiments, the TP53 gene or protein sequence is determined and anychange in the determined sequence relative to the wildtype sequence isdetected. In some embodiments, the TP53 gene sequence is determined byPCR, RT-PCR, Northern Blot. Southern Blot, and/or in situ hybridization.Another way to determine if TP53 contains a somatic mutation may involvethe use of an antibody-based detection assay (e.g. ELISA,immunohistochemistry, and/or Western Blot). In some embodiments, theantibody-based detection assay utilizes an antibody that binds to amutant TP53 protein with a tighter affinity than it binds to a wildtypeTP53 protein. The skilled artisan will also readily appreciate methodsof determining somatic mutations in TP53 based on the disclosures ofU.S. Pat. Nos. 5,843,654, 5,620,848, EP0390323 and U.S. Pat. No.5,527,676, all of which are herein incorporated by reference in theirentirely.

In some embodiments, the disclosure provides for a device useful fordetecting the methylation status of any of the informative loci, orfragments or complements thereof, disclosed herein. In some embodiments,the disclosure provides for a kit comprising components useful fordetecting the methylation status of the informative loci, or fragments,or complements thereof, disclosed herein. In some embodiments, the kitcomprises a swallowable balloon for collecting an esophageal sample fromthe subject. In some embodiments, the kit comprises any of theswallowable balloon devices disclosed in WO 2015/089422, which isincorporated herein in its entirety. In some embodiments, the disclosureprovides for a kit comprising primers for amplifying any of theinformative loci described herein, and instructions for performing anyof the methods disclosed herein. In some embodiments, the kit furthercomprises bisulfite. In some embodiments, the kit further comprises anobject suitable for collecting a sample from a subject (e.g., a brushand or balloon). In some embodiments, the disclosure provides for a kitcomprising any of the therapeutic agents disclosed herein andinstructions for performing any of the therapeutic methods disclosedherein.

In certain embodiments, the present disclosure provides drug screeningassays for identifying test compounds which potentiate the tumorsuppressor function of polypeptides encoded by sequences located in theinformative loci disclosed herein. In one aspect, the assays detect testcompounds which potentiate the expression level of polypeptides encodedby sequences located in the informative loci disclosed herein. Inanother aspect, the assays detect test compounds which inhibit themethylation of DNA. In certain embodiments, drug screening assays can begenerated which detect test compounds on the basis of their ability tointerfere with stability or function of polypeptides encoded bysequences located in the informative loci disclosed herein.

A variety of assay formats may be used and, in light of the presentdisclosure, those not expressly described herein will nevertheless beconsidered to be within the purview of ordinary skill in the art. Assayformats can approximate such conditions as protein expression level,methylation status of nucleotide sequences, tumor suppressing activity,and may be generated in many different forms. In many embodiments, thedisclosure provides assays including both cell-free systems andcell-based assays which utilize intact cells.

Compounds to be tested can be produced, for example, by bacteria, yeastor other organisms (e.g., natural products), produced chemically (e.g.,small molecules, including peptidomimetics), or produced recombinantly.The efficacy of the compound can be assessed by generating dose responsecurves from data obtained using various concentrations of the testcompound. Moreover, a control assay can also be performed to provide abaseline for comparison. In the control assay, the formation ofcomplexes is quantitated in the absence of the test compound.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays of the present invention which are performed in cell-freesystems, such as may be developed with purified or semi-purifiedproteins or with lysates, are often preferred as “primary” screens inthat they can be generated to permit rapid development and relativelyeasy detection of an alteration in a molecular target which is mediatedby a test compound. Moreover, the effects of cellular toxicity and/orbioavailability of the test compound can be generally ignored in the invitro system, the assay instead being focused primarily on the effect ofthe drug on the molecular target as may be manifest in an alteration ofbinding affinity with other proteins or changes in enzymatic propertiesof the molecular target.

In certain embodiments, test compounds identified from these assay s maybe used in a therapeutic method of treating cancer.

Still another aspect of the application provides transgenic non-humananimals which express a gene located within any one of the informativeloci disclosed herein, or which have had one or more of such genomicgene(s) disrupted in at least one of the tissue or cell-types of theanimal.

In another aspect, the application provides an animal model for cancer,which has a mis-expressed allele of a gene located within any one of theinformative loci disclosed herein. Such a mouse model can then be usedto study disorders arising from mis-expression of genes located withinany one of the informative loci disclosed herein.

Genetic techniques which allow for the expression of transgenes can beregulated via site-specific genetic manipulation in vivo are known tothose skilled in the art. For instance, genetic systems are availablewhich allow for the regulated expression of a recombinase that catalyzesthe genetic recombination a target sequence. As used herein, the phrase“target sequence” refers to a nucleotide sequence that is geneticallyrecombined by a recombinase. The target sequence is flanked byrecombinase recognition sequences and is generally either excised orinverted in cells expressing recombinase activity. Recombinase catalyzedrecombination events can be designed such that recombination of thetarget sequence results in either the activation or repression ofexpression of the polypeptides. For example, excision of a targetsequence which interferes with the expression of a recombinant gene canbe designed to activate expression of that gene. This interference withexpression of the protein can result from a variety of mechanisms, suchas spatial separation of the gene from the promoter element or aninternal stop codon. Moreover, the transgene can be made wherein thecoding sequence of the gene is flanked recombinase recognition sequencesand is initially transfected into cells in a 3′ to 5′ orientation withrespect to the promoter element. In such an instance, inversion of thetarget sequence will reorient the subject gene by placing the 5′ end ofthe coding sequence in an orientation with respect to the promoterelement which allow for promoter driven transcriptional activation.

In an illustrative embodiment, either the cre/loxP recombinase system ofbacteriophage P1 (Lakso et al., (1992) Proc. Nal. Acad. Sci. USA89:6232-6236; Orban et al., (1992) Proc. Natl. Acad Sci. USA89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al., (1991) Science 251:1351-1355; PCT publication WO92/15694) can be used to generate in vivo site-specific geneticrecombination systems. Cre recombinase catalyzes the site-specificrecombination of an intervening target sequence located between loxPsequences, loxP sequences are 34 base pair nucleotide repeat sequencesto which the Cre recombinase binds and are required for Cre recombinasemediated genetic recombination. The orientation of loxP sequencesdetermines whether the intervening target sequence is excised orinverted when Cre recombinase is present (Abremski et al., (1984) J.Biol. Chem. 259:1509-1514); catalyzing the excision of the targetsequence when the loxP sequences are oriented as direct repeats andcatalyzes inversion of the target sequence when loxP sequences areoriented as inverted repeats.

V. Subjects and Samples

In certain aspects, the invention relates to a subject suspected ofhaving or has: a cancer, a metaplasia, or a neoplasia of the uppergastrointestinal tract (e.g., esophageal cancer). Alternatively, asubject may be undergoing routine screening and may not necessarily besuspected of having such metaplasia or neoplasia In some embodiments,the subject is a human subject, and the neoplasia is a neoplasia of theupper gastrointestinal tract, such as the esophagus. In someembodiments, the subject is a human subject, and the metaplasia isBarrett's esophagus.

Assaying for biomarkers discussed above in a sample from subjects notknown to have, e.g., a metaplasia or neoplasia of the upper or lowergastrointestinal tract can aid in diagnosis of such a metaplasia orneoplasia in the subject. To illustrate, detecting the methylationstatus of the nucleotide sequences by MSP can be used by itself, or incombination with detecting the somatic mutation status of TP53 or othervarious assays, to improve the sensitivity and/or specificity fordetecting, e.g., a neoplasia of the upper or lower gastrointestinaltract. In some embodiments, such detection is made at an early stage inthe development of cancer, so that treatment is more likely to beeffective.

In some embodiments, an informative loci in a subject is considered“methylated” for the purposes of determining whether or not the subjectis prone to developing and/or has developed a metaplasia in theesophagus (e.g., Barrett's esophagus) or neoplasia (e.g., Barrett'sesophagus with dysplasia such as high-grade or low-grade dysplasia)(e.g., esophageal cancer such as esophageal adenocarcinoma) if the lociis at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%methylated. In some embodiments, a DNA sample from a subject is treatedwith bisulfite, and the resulting bisulfite sequence corresponds to anyof the nucleotide sequences disclosed herein comprising a “Y”nucleotide. In some embodiments, if at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 of the Y residues of the bisulfite-converted sequence havea C, the sequence is considered “methylated” for the purposes ofdetermining whether or not the subject is prone to developing and/or hasdeveloped a metaplasia in the esophagus (e.g., Barrett's esophagus) orneoplasia (e.g., Barrett's esophagus with dysplasia such as high-gradeor low-grade dysplasia) (e.g., esophageal cancer such as esophagealadenocarcinoma). In some embodiments, a DNA sample from a subject istreated with bisulfite, and the resulting bisulfite sequence correspondsto any of the nucleotide sequences disclosed herein comprising a “Y”nucleotide. In some embodiments, if at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% of the Y residues of the bisulfite-convertedsequence have a C, the sequence is considered “methylated” for thepurposes of determining whether or not the subject is prone todeveloping and/or has developed a metaplasia in the esophagus (e.g,Barrett's esophagus) or neoplasia (e.g., Barrett's esophagus withdysplasia such as high-grade or low-grade dysplasia) (e.g., esophagealcancer such as esophageal adenocarcinoma). In some embodiments, asubject is determined to be prone to developing and/or has developed ametaplasia in the esophagus (e.g., Barrett's esophagus) or neoplasia (eg., Barrett's esophagus with dysplasia such as high-grade or low-gradedysplasia) (e.g., esophageal cancer such as esophageal adenocarcinoma)if a certain number of “Y” nucleotides in a bisulfite converted sequenceare cytosines. In some embodiments, the certain number is at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 of the Y residues of thebisulfite-converted sequence. In some embodiments, the certain number isleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the Yresidues of the bisulfite-converted sequence. In certain embodiments, asubject is determined to be prone to developing and/or has developed ametaplasia in the esophagus (e.g., Barrett's esophagus) or neoplasia(e.g., esophageal cancer such as esophageal adenocarcinoma) (e.g.,Barrett's esophagus with dysplasia such as high-grade or low-gradedysplasia) if a certain percentage of DNA molecules from a sample from asubject are determined to be “methylated,” as defined herein. In someembodiments, the certain percentage of DNA molecules is at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the DNA moleculesfrom the sample are determined to be “methylated.” In some embodiments,the percentage of methylated DNA molecules is determined usingnext-generation sequencing. Exemplary cut-offs of DNA methylation andDNA molecule percentages may be found in the Examples section providedherein.

In particular embodiments, a vimentin sequence is considered“methylated” or a “methylated read” if at least 70%, 75%, 80%, 90% or100% of the CpG cytosines in a vimentin sequence, or portion thereof,are methylated. In some embodiments, if at least 8, 9 or 10 of the “Y”nucleotides in a portion of a vimentin sequence (such as the sequence ofSEQ ID NOs: 16207 and/or 16208, or a portion thereof, such as thesequence of SEQ ID NOs: 16209 and/or 16210) have a C, then the vimentinsequence is considered “methylated” or a “read.” In some embodiments,the primers used to amplify the portion of the vimentin nucleic acidsequence comprise the nucleotide sequences of SEQ ID NOs: 16209 and/or16210. In some embodiments, if at least 0.5%, 0.8%, 0.9%, 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, or 9% of the vimentin nucleic acid sequences in asample from a subject are considered methylated or a read, then thesubject is determined to have an esophageal metaplasia and/or neoplasia.In particular embodiments, if at least 1% (e.g., at least 1.05%) of thevimentin nucleic acid sequences in a sample from a subject areconsidered methylated or a read, then the subject is determined to havean esophageal metaplasia and/or neoplasia.

In particular embodiments, a SqBE18 sequence is considered “methylated”or a “methylated read” if at least 65%, 70%, 75%, 80%, 90% or 100% ofthe CpG cytosines in a SqBE18 sequence (e.g., a nucleotide sequence atleast 80%, 85%, 901%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identity to the sequence of SEQ ID NO: 8220, 8262, 8304 or 8346),or a portion thereof, are methylated. In some embodiments, if at least0.5%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% of the SqBE18nucleic acid sequences in a sample from a subject are consideredmethylated or a read, then the subject is determined to have anesophageal metaplasia and/or neoplasia. In particular embodiments, if atleast 2%, 2.5%, or 3% (e.g., at least 3.11%) of the SqBE18 nucleic acidsequences in a brushing sample from a subject are considered methylatedor a read, then the subject is determined to have an esophagealmetaplasia and/or neoplasia In particular embodiments, if at least 1% ofthe SqBE18 nucleic acid sequences in a balloon sample from a subject areconsidered methylated or a read, then the subject is determined to havean esophageal metaplasia and/or neoplasia.

In some embodiments, the disclosure provides for a method of diagnosingwhether a subject has an esophageal neoplasia or metaplasia, comprising:obtaining a sample from a subject; measuring the amount of methylatedcytosines in CpG dinucleotides in a vimentin nucleic acid sequence, orportion thereof, obtained from the sample; wherein if at least 65%, atleast 70%, at least 75%, or preferably at least 80% of the cytosines inCpG dinucleotides in the vimentin nucleic acid sequence, or portionthereof, are methylated, than the vimentin nucleic acid sequence, orportion thereof, is considered a read; and measuring the number of readspresent in the sample; wherein if at least 1% of the vimentin nucleicacid sequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, the vimentin nucleic acid sequences from the sampleare treated with bisulfite. In some embodiments, the sequence of thebisulfite converted nucleic acid sequences is determined bynext-generation sequencing. In some embodiments, the level of methylatedcytosines is determined in an amplified portion of the bisulfiteconverted vimentin nucleic acid sequence obtained from the subject Insome embodiments, the amplified portion comprises 10 CpGs. In someembodiments, the primers used to amplify the portion of the vimentinnucleic acid sequence comprise SEQ ID NOs: 16209 and 16210. In someembodiments, the amplified portion comprises the nucleotide sequence ofSEQ ID NOs: 16207 and/or 16208, and the region between the amplificationprimers comprises the nucleotide sequences of SEQ ID Nos: 16209 and/or162010. In some embodiments, if at least 0.5% to 5% of the vimentinnucleic acid sequences, or portions thereof, in the sample are reads,than the subject is determined to have an esophageal neoplasia ormetaplasia In some embodiments, if at least 0.5% to 3% of the vimentinnucleic acid sequences, or portions thereof, in the sample are reads,than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if at least 0.5% to 1.5% of thevimentin nucleic acid sequences, or portions thereof, in the sample arereads, than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if at least 0.95% to 1.16% of thevimentin nucleic acid sequences, or portions thereof, in the sample arereads, than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if at least 1.02% of the vimentinnucleic acid sequences, or portions thereof, in the sample are reads,than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if at least 3% of the vimentin nucleicacid sequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if at least 5% of the vimentin nucleic acid sequences,or portions thereof, in the sample are reads, than the subject isdetermined to have an esophageal neoplasia or metaplasia. In someembodiments, if the sample is obtained from a brushing, and if at least0.5% to 1.5% of the vimentin nucleic acid sequences, or portionsthereof, in the sample are reads, than the subject is determined to havean esophageal neoplasia or metaplasia. In some embodiments, if thesample is obtained from a brushing, and if at least 1.05% of thevimentin nucleic acid sequences, or portions thereof, in the sample arereads, than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if the sample is obtained from aballoon, and if at least 0.5% to 1.5% of the vimentin nucleic acidsequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if the sample is obtained from a balloon, and if atleast 0.95% to 1.16% of the vimentin nucleic acid sequences, or portionsthereof, in the sample are reads, than the subject is determined to havean esophageal neoplasia or metaplasia. In some embodiments, if thesample is obtained from a balloon, and if at least 1% of the vimentinnucleic acid sequences, or portions thereof, in the sample are reads,than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if the subject is determined to have anesophageal neoplasia or metaplasia, then the subject may be administeredany of cryotherapy, photodynamic therapy (PDT); radiofrequency ablation(RFA); laser ablation; argon plasma coagulation (APC);electrocoagulation (electrofulguration); esophageal stent, surgery,and/or a therapeutic agent.

In some embodiments, the disclosure provides for a method of diagnosingwhether a subject has an esophageal neoplasia or metaplasia, comprising:obtaining a sample from a subject; measuring the amount of methylatedcytosines in CpG dinucleotides in a SqBE18 nucleic acid sequence, orportion thereof, obtained from the sample, wherein if at least 65%, atleast 70%, or at least 75% (e.g., at least 71% or at least 76%) of thecytosines in CpG dinucleotides in the SqBE18 nucleic acid sequence, orportion thereof, are methylated, than the SqBE18 nucleic acid sequence,or portion thereof, is considered a read; and measuring the number ofreads present in the sample; wherein if at least 0.5%, at least 1%, atleast 2%, or at least 3% (e.g. least 3.11%) of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, the SqBE18 nucleic acid sequences from the sample aretreated with bisulfite. In some embodiments, the sequence of thebisulfite converted nucleic acid sequences is determined bynext-generation sequencing. In some embodiments, the level of methylatedcytosines is determined in an amplified portion of the bisulfiteconverted SqBE18 nucleic acid sequence obtained from the subject. Insome embodiments, if at least 0.5% to 5% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if at least 0.5% to 3.5% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if at least 1% to 3.11% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if at least 0.76% to 1.06% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if at least 3.11% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if at least 0.1% of the SqBE18 nucleic acid sequences,or portions thereof, in the sample are reads, than the subject isdetermined to have an esophageal neoplasia or metaplasia In someembodiments, if at least 1% of the SqBE18 nucleic acid sequences, orportions thereof, in the sample are reads, than the subject isdetermined to have an esophageal neoplasia or metaplasia. In someembodiments, if the sample is obtained from a brushing, and if at least2% to 3.11% of the SqBE18 nucleic acid sequences, or portions thereof,in the sample are reads, than the subject is determined to have anesophageal neoplasia or metaplasia. In some embodiments, if the sampleis obtained from a brushing, and if at least 3.11% of the SqBE18 nucleicacid sequences, or portions thereof, in the sample are reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if the sample is obtained from a balloon, and if atleast 0.5% to 1.5% of the SqBE18 nucleic acid sequences, or portionsthereof, in the sample are reads, than the subject is determined to havean esophageal neoplasia or metaplasia. In some embodiments, if thesample is obtained from a balloon, and if at least 0.76% to 1.06% of theSqBE18 nucleic acid sequences, or portions thereof, in the sample arereads, than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if the sample is obtained from aballoon, and if at least 1% of the SqBE18 nucleic acid sequences, orportions thereof, in the sample are reads, than the subject isdetermined to have an esophageal neoplasia or metaplasia. In someembodiments, if the subject is determined to have an esophagealneoplasia or metaplasia, then the subject may be administered any ofcryotherapy, photodynamic therapy (PDT); radiofrequency ablation (RFA);laser ablation; argon plasma coagulation (APC); electrocoagulation(electrofulguration); esophageal stent, surgery, and/or a therapeuticagent.

In some embodiments, the disclosure provides for a method of diagnosingwhether a subject has an esophageal neoplasia or metaplasia, comprising:obtaining a sample from a subject; measuring the amount of methylatedcytosines in CpG dinucleotides in a vimentin nucleic acid sequence andin a SqBE18 nucleic acid sequence, or portions thereof, obtained fromthe sample; wherein if at least 65%, at least 70%, at least 75%, orpreferably at least 80% of the cytosines in CpG dinucleotides in thevimentin nucleic acid sequence, or portion thereof, are methylated, thanthe vimentin nucleic acid sequence, or portion thereof, is considered aread for VIM (vimentin); wherein if at least 65%, at least 70%, or atleast 75% (e.g., at least 71% or at least 76%) of the cytosines in CpGdinucleotides in the SqBE18 nucleic acid sequence, or portion thereof,are methylated, than the SqBE18 nucleic acid sequence, or portionthereof, is considered a read for SqBE18; and measuring the number ofVIM and SqBE18 reads present in the sample; wherein if at least 1% ofthe vimentin nucleic acid sequences, or portions thereof, in the sampleare VIM reads and if at least 0.5%, at least 1%, at least 2%, or atleast 3% (e.g. least 3.11%) of the SqBE18 nucleic acid sequences, orportions thereof, in the sample are SqBE18 reads, than the subject isdetermined to have an esophageal neoplasia or metaplasia. In someembodiments, the vimentin or SqBE18 nucleic acid sequences from thesample are treated with bisulfite. In some embodiments, the sequence ofthe bisulfite converted nucleic acid sequences is determined bynext-generation sequencing. In some embodiments, the level of methylatedcytosines is determined in an amplified portion of the bisulfiteconverted vimentin or SqBE18 nucleic acid sequence obtained from thesubject. In some embodiments, the amplified vimentin portion comprises10 CpGs. In some embodiments, the amplified SqBE18 portion comprises 21CpGs. In some embodiments, the primers used to amplify the portion ofthe bisulfite converted vimentin nucleic acid sequence comprise SEQ IDNOs: 16209 and 16210. In some embodiments, the amplified portioncomprises the nucleotide sequence of SEQ ID NOs: 16207 and/or 16208, andthe region between the amplification primers comprises the nucleotidesequences of SEQ ID Nos: 16209 and/or 16210. In some embodiments, theprimers used to amplify the portion of the bisulfite converted SqBE18nucleic acid sequence comprise SEQ ID NOs: 8388 and 8402. In someembodiments, the amplified portion comprises the nucleotide sequence ofSEQ ID NOs: 8318 and/or 8360, or fragments and/or reverse complementsthereof. In some embodiments, the amplified portion comprises thenucleotide sequences of SEQ ID NOs: 8332 and/or 8374, or fragmentsand/or reverse complements thereof, which may be generated from fullymethylated parental templates. In some embodiments, if at least 0.5% to5% of the vimentin nucleic acid sequences, or portions thereof, in thesample are VIM reads, and if at least 0.5% to 5% of the SqBE18 nucleicacid sequences, or portions thereof, in the sample are SqBE18 reads,than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if at least 0.5% to 3% of the vimentinnucleic acid sequences, or portions thereof, in the sample are VIMreads, and if at least 0.5% to 3.5% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are SqBE18 reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if at least 0.5% to 1.5% of the vimentin nucleic acidsequences, or portions thereof, in the sample are VIM reads, and if atleast 1% to 3.11% of the SqBE18 nucleic acid sequences, or portionsthereof, in the sample are SqBE18 reads, than the subject is determinedto have an esophageal neoplasia or metaplasia. In some embodiments, ifat least 1% of the vimentin nucleic acid sequences, or portions thereof,in the sample are VIM reads, and if at least 1% of the SqBE18 nucleicacid sequences, or portions thereof, in the sample are SqBE18 reads,than the subject is determined to have an esophageal neoplasia ormetaplasia In some embodiments, if at least 0.5% of the vimentin nucleicacid sequences, or portions thereof, in the sample are VIM reads, and ifat least 3.11% of the SqBE18 nucleic acid sequences, or portionsthereof, in the sample are SqBE18 reads, than the subject is determinedto have an esophageal neoplasia or metaplasia. In some embodiments, ifthe sample is obtained from a brushing, and if at least 0.5% to 1.5% ofthe vimentin nucleic acid sequences, or portions thereof, in the sampleare VIM reads, and if at least 2% to 3.11% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are SqBE18 reads, than thesubject is determined to have an esophageal neoplasia or metaplasia. Insome embodiments, if the sample is obtained from a brushing, and if atleast 1.05% of the vimentin nucleic acid sequences, or portions thereof,in the sample are VIM reads, and if at least 3.11% of the SqBE18 nucleicacid sequences, or portions thereof, in the sample are SqBE18 reads,than the subject is determined to have an esophageal neoplasia ormetaplasia In some embodiments, if the sample is obtained using aballoon, and if at least 0.5% to 1.5% of the vimentin nucleic acidsequences, or portions thereof, in the sample are VIM reads, and if atleast 0.5% to 1.5% of the SqBE18 nucleic acid sequences, or portionsthereof, in the sample are SqBE18 reads, than the subject is determinedto have an esophageal neoplasia or metaplasia. In some embodiments, ifthe sample is obtained using a balloon, and if at least 0.95% to 1.16%of the vimentin nucleic acid sequences, or portions thereof in thesample are reads, and if at least 0.76% to 1.06% of the SqBE18 nucleicacid sequences, or portions thereof, in the sample are SqBE18 reads,than the subject is determined to have an esophageal neoplasia ormetaplasia. In some embodiments, if the sample is obtained from aballoon, and if at least 1% of the vimentin nucleic acid sequences, orportions thereof, in the sample are reads, and if at least 1% of theSqBE18 nucleic acid sequences, or portions thereof, in the sample areSqBE18 reads, than the subject is determined to have an esophagealneoplasia or metaplasia. In some embodiments, if the subject isdetermined to have an esophageal neoplasia or metaplasia, then thesubject may be administered any of cryotherapy, photodynamic therapy(PDT), radiofrequency ablation (RFA); laser ablation; argon plasmacoagulation (APC); electrocoagulation (electrofulguration); esophagealstent, surgery, and/or a therapeutic agent.

In addition to diagnosis, assaying of a marker in a sample from asubject not known to have, e.g., a metaplasia or neoplasia of the uppergastrointestinal tract, can be prognostic for the subject (i e.,indicating the probable course of the disease). To illustrate, subjectshaving a predisposition to develop a metaplasia or neoplasia of theupper gastrointestinal tract may possess methylated nucleotidesequences. Assaying of methylated informative loci (e.g., vimentinand/or SqBE18) in a sample from subjects either by itself, or incombination with assaying for somatic mutation(s) in TP53, can also beused to select a particular therapy or therapies which are particularlyeffective against, e.g., a neoplasia or metaplasia of the uppergastrointestinal tract in the subject, or to exclude therapies that arenot likely to be effective.

Assaying of methylated informative loci (e.g., vimentin and/or SqBE18)in samples from subjects that are known to have, or to have had, acancer is also useful. For example, the present methods can be used toidentify whether therapy is effective or not for certain subjects. Oneor more samples are taken from the same subject prior to and followingtherapy, and assayed for any one or more of the informative loci markerseither by itself or themselves, or in combination with assaying forsomatic mutation(s) in TP53. A finding that an informative locus ismethylated in the sample taken prior to therapy and absent (or at alower level) after therapy may indicate that the therapy is effectiveand need not be altered. In those cases where the informative locus ismethylated in the sample taken before therapy and in the sample takenafter therapy, it may be desirable to alter the therapy to increase thelikelihood that the cancer will be reduced in the subject. Thus, thepresent method may obviate the need to perform more invasive procedureswhich are used to determine a patient's response to therapy.

Cancers frequently recur following therapy in patients with advancedcancers. In this and other instances, the assays of the invention areuseful for monitoring over time the status of a cancer associated withsilencing of genes located in the informative loci disclosed herein. Forsubjects in whom a cancer is progressing, there can be no DNAmethylation in some or all samples when the first sample is taken andthen appear in one or more samples when the second sample is taken. Forsubjects in which cancer is regressing, DNA methylation may be presentin one or a number of samples when the first sample is taken and then beabsent in some or all of these samples when the second sample is taken.

Samples for use with the methods described herein may be essentially anybiological material of interest, e.g., a collection of cells taken froma subject. For example, a sample may be a bodily fluid sample from asubject, a tissue sample from a subject, a solid or semi-solid samplefrom a subject, a primary cell culture or tissue culture of materialsderived from a subject, cells from a cell line, or medium or otherextracellular material from a cell or tissue culture, or a xenograft(meaning a sample of a cancer from a first subject, e.g., a human, thathas been cultured in a second subject, e.g., an immuno-compromisedmouse). The term “sample” as used herein is intended to encompass both abiological material obtained directly from a subject (which may bedescribed as the primary sample) as well as any manipulated forms orportions of a primary sample. A sample may also be obtained bycontacting a biological material with an exogenous liquid, resulting inthe production of a lavage liquid containing some portion of thecontacted biological material. Furthermore, the term “sample” isintended to encompass the primary sample after it has been mixed withone or more additive, such as preservatives, chelators, anti-clottingfactors, etc. In some embodiments, a sample is obtained by means of acytology brushing and/or a balloon. In some embodiments, the sample isobtained from a subject's gastroesophageal junction.

In certain embodiments, a bodily fluid sample is a blood sample In thiscase, the term “sample” is intended to encompass not only the blood asobtained directly from the patient but also fractions of the blood, suchas plasma, serum, cell fractions (e.g., platelets, erythrocytes, andlymphocytes), protein preparations, nucleic acid preparations, etc Insome embodiments, the bodily fluid may be derived from the stomach, forexample, gastric secretions, acid reflux, or vomit. In otherembodiments, the bodily fluid may be a fluid secreted by the pancreas orbladder. In other embodiments, the body fluid may be saliva, spit, or anesophageal washing. In certain embodiments, a tissue sample is a biopsytaken from the mucosa of the gastrointestinal tract. In otherembodiments, a tissue sample is the brushings from, e.g., the esophagusof a subject.

A subject is in some embodiments a human subject, but it is expectedthat the molecular markers disclosed herein, and particularly theirhomologs from other animals, are of similar utility in other animals. Incertain embodiments, it may be possible to detect a biomarker describedherein (e.g., DNA methylation or protein expression level) directly inan organism without obtaining a separate portion of biological material.In such instances, the term “sample” is intended to encompass thatportion of biological material that is contacted with a reagent ordevice involved in the detection process.

In certain embodiments, DNA which is used as the template is obtainedfrom a bodily fluid sample. Examples of bodily fluids are blood, saliva,spit or an esophageal washing. Other body fluids can also be used.Because they can be easily obtained from a subject and can be used toscreen for multiple diseases, blood or blood-derived fractions areespecially useful. Blood-derived fractions can comprise blood, serum,plasma, or other fractions. For example, a cellular fraction can beprepared as a “buffy coat” (i.e., leukocyte-enriched blood portion) bycentrifuging 5 ml of whole blood for 10 min at 800 times gravity at roomtemperature. Red blood cells sediment most rapidly and are present asthe bottom-most fraction in the centrifuge tube. The buffy coat ispresent as a thin creamy white colored layer on top of the red bloodcells. The plasma portion of the blood forms a layer above the buffycoat. Fractions from blood can also be isolated in a variety of otherways. One method is by taking a fraction or fractions from a gradientused in centrifugation to enrich for a specific size or density ofcells.

In some embodiments, DNA is isolated from samples. Procedures forisolation of DNA from such samples are well known to those skilled inthe art. Commonly, such DNA isolation procedures comprise lysis of anycells present in the samples using detergents, for example. After celllysis, proteins are commonly removed from the DNA using variousproteases. RNA is removed using RNase. The DNA is then commonlyextracted with phenol, precipitated in alcohol and dissolved in anaqueous solution.

VI. Therapeutic Methods

In some embodiments, the disclosure provides for a method of determiningwhether a subject has any one or more of the methylated informative locidisclosed herein (e.g., vimentin and/or SqBE18) that are indicative ofthe presence of a metaplasia in the esophagus (e.g., Barrett'sesophagus), wherein if the subject is determined to have a metaplasia inthe esophagus (e.g., Barrett's esophagus), the subject is treated withan agent that treats the metaplasia in the esophagus (e.g., Barret'sesophagus). In some embodiments, the disclosure provides for a method oftreating a subject determined to have a metaplasia m the esophagus(e.g., Barrett's esophagus). In some embodiments, the treatment of ametaplasia in the esophagus (e.g., Barrett's esophagus) encompassesadministration of any one or more of the following compounds: protonpump inhibitors (PPIs) such as omeprazole (Prilosec, Zegerid),lansoprazolc (Prevacid), pantoprazole (Protonix), rabeprazole (AcipHex),esomeprazole (Nexium), dexlansoprazole (Dexilant). Histamine H2 receptorblocking agents such as cimetidine (Tagamet), ranitidine (Zantac),famotidine (Pepcid) and nizatidine (Axid). Turns, Rolaids, or otherquick-acting reflux medications. Prokinetic agents, or drugs that helpmove food through the gastrointestinal tract more quickly, offer anattractive alternative either alone or in combination with acidinhibition. In some embodiments, the treatment of a metaplasia in theesophagus (e.g., Barrett's esophagus) is endoscopic mucosal resection(EMR); photodynammic therapy (PDT); radiofrequency ablation (RFA); argonplasma coagulation (APC); cryotherapy, and/or surgery (e.g.esophagectomy, anti-reflux surgery).

In some embodiments, the disclosure provides for a method of determiningwhether a subject has any one or more of the methylated informative locidisclosed herein that are indicative of esophageal neoplasia (e.g.,esophageal cancer), wherein if the subject is determined to have anesophageal neoplasia (e.g., esophageal cancer), the subject is treatedwith an agent that treats the esophageal neoplasia (e.g., esophagealcancer) In some embodiments, the disclosure provides for a method ofdetermining whether a subject has any one or more of the methylatedinformative loci disclosed herein in combination with any of the TP53somatic mutations disclosed herein that are indicative of esophagealneoplasia (e.g., esophageal cancer), wherein if the subject isdetermined to have an esophageal neoplasia (e.g., esophageal cancer),the subject is treated with an agent that treats the esophagealneoplasia (e.g., esophageal cancer). In some embodiments, the disclosureprovides for a method of treating a subject determined to haveesophageal neoplasia (e.g., esophageal cancer). In some embodiments, theesophageal neoplasia is Barrett's esophagus with low grade dysplasia,Barrett's esophagus with high grade dysplasia (HGD) and/or esophagealadenocarcinoma (EAC). In some embodiments, the treatment of esophagealneoplasia (e.g., esophageal cancer) encompasses surgery (e.g.esophagectomy), radiation therapy, chemoradiation therapy and/orchemotherapy. In some embodiments, the treatment of esophageal neoplasia(e.g., esophageal cancer) encompasses administering one or morechemotherapeutic agent, such as any one or more therapeutic agentselected from the group consisting of: carboplatin and paclitaxel(Taxol®) (which may be combined with radiation); cisplatin and5-fluorouracil (5-FU) (often combined with radiation); ECF: epirubicine(Ellence®), cisplatin, and 5-FU (especially for gastroesophagealjunction tumors); DCF: docetaxel (Taxotere®), cisplatin, and 5-FU;Cisplatin with capecitabine (Xeloda®); oxaliplatin and either 5-FU orcapecitabine; doxorubicin (Adriamycin®), bleomycin, mitomycin,methotrexate, vinorelbine (Navelbine®), topotecan, and irinotecan(Camptosar®). In some embodiments, for some esophagus cancers thatoverexpress the HER2 protein on the surface of their cells, chemotherapymay be used along with the targeted drug trastuzumab. Ramucirumab may beused to treat cancers that start at the gastroesophageal (GE) junctionwhen they are advanced. In some embodiments, the treatment encompassesendoscopic treatments, such as endoscopic mucosal resection (EMR)followed by treatment with proton pump inhibitors, photodynamic therapy(PDT); radiofrequency ablation (RFA); laser ablation; argon plasmacoagulation (APC); electrocoagulation (electrofulguration); oresophageal stent.

The terms “treatment”, “treating”, “alleviation” and the like are usedherein to generally mean obtaining a desired pharmacologic and/orphysiologic effect, and may also be used to refer to improving,alleviating, and/or decreasing the severity of one or more symptoms of acondition being treated. The effect may be prophylactic in terms ofcompletely or partially delaying the onset or recurrence of a disease,condition, or symptoms thereof, and/or may be therapeutic in terms of apartial or complete cure for a disease or condition and/or adverseeffect attributable to the disease or condition. “Treatment” as usedherein covers any treatment of a disease or condition of a mammal,particularly a human, and includes: (a) preventing the disease orcondition from occurring in a subject which may be predisposed to thedisease or condition but has not yet been diagnosed as having it; (b)inhibiting the disease or condition (e.g., arresting its development);or (c) relieving the disease or condition (e.g., causing regression ofthe disease or condition, providing improvement in one or moresymptoms).

Treating a metaplasia (e.g., Barrett's esophagus) and/or a neoplasia(e.g., esophageal cancer) in a subject refers to improving (improvingthe subject's condition), alleviating, delaying or slowing progressionor onset, decreasing the severity of one or more symptoms associatedwith a metaplasia (e.g., Barrett's esophagus) and/or a neoplasia (e.g.,esophageal cancer). For example, treating a metaplasia or neoplasiaincludes any one or more of: reducing growth, proliferation and/orsurvival of metaplastic/neoplastic cells, killing metaplastic/neoplasticcells (e.g., by necrosis, apoptosis or autophagy), decreasingmetaplasia/neoplasia size, decreasing rate of metaplasia/neoplasia sizeincrease, halting increase in metaplasia/neoplasia size, improvingability to swallow, decreasing internal bleeding, decreasing incidenceof vomiting, reducing fatigue, decreasing the number of metastases,decreasing pain, increasing survival, and increasing progression freesurvival.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1: Identification of Esophageal Cancer Informative Loci

Methylated informative loci were initially identified using thetechnique of reduced representation bisulfite sequencing (RRBS) in adiscovery set of 23 paired biopsies of normal squamous esophagus andmatched esophageal adenocarcinomas, along with biopsies of 8 Barrett'sesophagus tissue, and along with brushings of 8 Barrett's esophagustissues (one BE brushing case also having a matched biopsy).

Discovery data were initially analyzed for each individual CpG residuein the RRBS data set. Individual CpGs were considered methylated in EACif they showed methylation in less than 10% of DNA sequence reads in allof the informative squamous samples, where at least 4 squamous sampleswere informative, where an informative sample had equal to or greaterthan 20 reads covering the CpG, and if 8 or more of the informative EACsamples demonstrated percent methylation at a level that was at least 20percentage points greater than the methylation level of the mostmethylated normal squamous sample. CpGs were similarly defined asmethylated in Barrett's esophagus if they showed methylation of lessthan 10% of DNA sequence reads of all informative squamous samples,where an informative sample had equal to or greater than 20 readscovering the CpG, and if 3 or more of the informative BE samplesdemonstrated percent methylation at a level that was at least 20percentage points greater than the methylation level of the mostmethylated normal squamous sample. CpGs meeting criteria for methylationin both EACs and BE were defined as methylated in both EAC and BE. Suchmethylated CpGs were then aggregated into patches by grouping togethermethylated CpGs that were within 200 bp of one another. Patches mayconsist of 1 CpG up to any number of CpGs that meet the above criteria.

The names assigned to 428 genomic patches defined as methylated in bothEAC and BE by the criteria above were recorded, and the sequences ofthese loci correspond to SEQ ID NOs: 1-856. The genomic coordinates ofthe genomic patches defined as methylated by the above criteria werealso recorded. The genomic sequences of these patches on the respectivegenomic (+) and (−) strands were determined and recorded. (Upper andlower case designations were used according to those of the UCSCbrowser, where lower case sequences are lower complexity DNA sequences).The bisulfite converted sequences of these corresponding patches (i.e,the bisulfite converted sequence of the (+) strand and the bisulfiteconverted sequence of the (−) strand) were determined and recorded (seesequences of SEQ ID NOs: 857-1281 and 1713-2140 for the bisulfiteconverted sequences of the respective (+) and (−) strands). C residuesthat may be methylated or unmethylated, and hence may be bisulfiteconverted to T (if unmethylated) or remain as a C (if methylated), weredesignated with a Y (where Y denotes C or T), and where, after bisulfiteconversion, actual maintenance of a Y designated base as a C was scoredas methylation at that base. Thus, these sequences represent the groupof all combinations of all sequences in which 0, 1, or more than one Yis converted to a T. The reverse complements of the bisulfite convertedsequences of the (+) and (−) strands will be obvious to one of ordinaryskill in the art and are also included by implication in thisdisclosure. The bisulfite converted sequences of the fully methylatedform of the corresponding patches (i.e, in which all Y bases in everybisulfite converted sequence are retained as a C), corresponding to the(+) strand the (−) strand were determined and recorded (see sequences ofSEQ ID NOs: 1285-1712 and 2141-2568 for the bisulfite convertedsequences of the fully methylated form of the (+) and (−) strandsrespectively of the corresponding patches). The reverse complements ofthe bisulfite converted methylated (+) stand and (−) stand sequenceswill be obvious to one of ordinary skill in the art and are alsoincluded by implication in this disclosure.

Patches were expanded by 100 base pairs on either side so as toaccommodate either the design of amplification primers or to excludeadditional presumptively methylated bases. The sequences of theseexpanded patches correspond to SEQ ID NOs: 2569-3424 and their genomiccoordinates were also recorded. The genomic sequences of these expandedpatches on the respective genomic (+) and (−) strands were determinedand recorded. (Upper and lower case designations were used according tothose of the UCSC browser, where lower case sequences are lowercomplexity DNA sequences) The bisulfite converted sequences of thesecorresponding expanded patches (i.e, the bisulfite converted sequence ofthe (+) strand and the bisulfite converted sequence of the (−) strand)were determined and recorded (see sequence of SEQ ID NOs: 3425-3852 and4281-4708 for the bisulfite converted sequences of the respective (+)and (−) strands). C residues that may be methylated or unmethylated, andhence may be bisulfite converted to T (if unmethylated) or remain as a C(if methylated), were designated with a Y (where Y denotes C or T), andwhere, after bisulfite conversion, actual maintenance of a Y designatedbase as a C was scored as methylation at that base. Thus, thesesequences represent the group of all combinations of all sequences inwhich 0, 1, or more than one Y is converted to a T. The reversecomplements of the bisulfite converted sequences of the (+) and (−)strands will be obvious to one of ordinary skill in the art and are alsoincluded by implication in this disclosure. The bisulfite convertedsequences of the fully methylated form of the corresponding expandedpatches (i.e, in which all Y bases in every bisulfite converted sequenceare retained as a C), corresponding to the (+) strand the (−) strandwere determined and recorded (see sequences of SEQ ID NOs: 3853-4280 and4789-5136 for the bisulfite converted sequences of the fully methylatedform of the (+) and (−) strands respectively of the correspondingexpanded patches). The reverse complements of the bisulfite convertedmethylated (+) stand and (−) stand sequences will be obvious to one ofordinary skill in the art and are also included by implication in thisdisclosure.

CpG islands overlapping patches that may contain additional CpGs thatare methylated coordinately with patches were also defined. Thesequences of these CpG islands correspond to SEQ ID NOs: 5137-5926. Thegenomic coordinates of the CpG islands were also recorded. The genomicsequences of these expanded patches on the respective genomic (+) and(−) strands were determined and recorded. (Upper and lower casedesignations were used according to those of the UCSC browser, wherelower case sequences are lower complexity DNA sequences). The bisulfiteconverted sequences of these corresponding CpG islands (i.e, thebisulfite converted sequence of the (+) strand and the bisulfiteconverted sequence of the (−) strand) were determined and recorded (seesequences of SEQ ID NOs: 5927-6321 and 6717-7111 for the bisulfiteconverted sequences of the respective (+) and (−) strands). C residuesthat may be methylated or unmethylated, and hence may be bisulfiteconverted to T (if unmethylated) or remain as a C (if methylated), weredesignated with a Y (where Y denotes C or T), and where, after bisulfiteconversion, actual maintenance of a Y designated base as a C was scoredas methylation at that base. Thus, these sequences represent the groupof all combinations of all sequences in which 0, 1, or more than one Yis converted to a T. The reverse complements of the bisulfite convertedsequences of the (+) and (−) strands will be obvious to one of ordinaryskill in the art and are also included by implication in thisdisclosure. The bisulfite converted sequences of the fully methylatedform of the corresponding CpG islands (i.e, in which all Y bases inevery bisulfite converted sequence are retained as a C), correspondingto the (+) strand the (−) strand were determined and recorded (seesequences of SEQ ID NOs: 6322-6716 and 7112-7506 for the bisulfiteconverted sequences of the fully methylated form of the (+) and (−)strands respectively of the corresponding CpG islands). The reversecomplements of the bisulfite converted methylated (+) stand and (−)stand sequences will be obvious to one of ordinary skill in the art andare also included by implication in this disclosure.

Regions of Interest (ROI) were defined that provided preferred regionsfor design of PCR amplicons that would encompass preferred patches. Thegenomic coordinates of the ROT were also recorded. The sequences of the(+) strands of the Regions of Interest correspond to SEQ ID NOs:8209-8222, and the sequences of the (−) strands Regions of Interestcorrespond to SEQ ID NOs: 8251-8261. (Upper and lower case designationswere used according to those of the UCSC browser, where lower casesequences are lower complexity DNA sequences). The bisulfite convertedsequences of these corresponding Regions of Interest (i.e, the bisulfiteconverted sequence of the (+) strand and the bisulfite convertedsequence of the (−) strand) were determined and recorded (see sequencesof SEQ ID NOs: 8223-8236 and 8265-8278 for the bisulfite convertedsequences of the respective (+) and (−) strands). C residues that may bemethylated or unmethylated, and hence may be bisulfite converted to T(if unmethylated) or remain as a C (if methylated), were designated witha Y (where Y denotes C or T), and where, after bisulfite conversion,actual maintenance of a Y designated base as a C was scored asmethylation at that base. Thus, these sequences represent the group ofall combinations of all sequences in which 0, 1, or more than one Y isconverted to a T. The reverse complements of the bisulfite convertedsequences of the (+) and (−) strands will be obvious to one of ordinaryskill in the art and are also included by implication in thisdisclosure. The bisulfite converted sequences of the fully methylatedform of the Regions of Interest (i.e, in which all Y bases in everybisulfite converted sequence are retained as a C), corresponding to the(+) strand the (−) strand were determined and recorded (see sequences ofSEQ ID NOs: 8237-8250 and 8279-8292 for the bisulfite convertedsequences of the fully methylated form of the (+) and (−) strandsrespectively of the corresponding Regions of Interest). The reversecomplements of the bisulfite converted methylated (+) stand and (−)stand sequences will be obvious to one of ordinary skill in the art andare also included by implication in this disclosure.

Specific PCR Amplicons were defined within the Regions of Interest(ROT). The genomic coordinates of the Amplicons were recorded. Thesequences of the (+) strands of the Amplicons correspond to SEQ ID NOs:8293-8306 and 8405-8409, and the sequences of the (−) strands of theAmplicons correspond to SEQ ID NOs: 8335-8348 and 8420-8424. (Upper andlower case designations were used according to those of the UCSCbrowser, where lower case sequences are lower complexity DNA sequences).The bisulfite converted sequences of these Amplicons (i.e, the bisulfiteconverted sequence of the (+) strand and the bisulfite convertedsequence of the (−) strand) were determined and recorded (see sequencesof SEQ ID NOs: 8307-8320 and 8410-8414 for the bisulfite convertedsequences of the (+) strands and see sequences of SEQ ID NOs: 8349-8362and 8425-8429 and for the bisulfite converted sequences of the (−)strands). C residues that may be methylated or unmethylated, and hencemay be bisulfite converted to T (if unmethylated) or remain as a C (ifmethylated), were designated with a Y (where Y denotes C or T), andwhere, after bisulfite conversion, actual maintenance of a Y designatedbase as a C was scored as methylation at that base. Thus, thesesequences represent the group of all combinations of all sequences inwhich 0, 1, or more than one Y is converted to a T. The reversecomplements of the bisulfite converted sequences of the (+) and (−)strands will be obvious to one of ordinary skill in the art and are alsoincluded by implication in this disclosure. The bisulfite convertedsequences of the fully methylated form of the Amplicons (i.e, in whichall Y bases in every bisulfite converted sequence are retained as a C),corresponding to the (−) strand the (−) strand were determined andrecorded (see sequences of SEQ ID NOs: 8321-8334 and 8415-8419 for thebisulfite converted sequences of the fully methylated form of the (+)strands and see sequences of SEQ ID NOs: 8363-8376 and 8430-8434 for thebisulfite converted sequences of the fully methylated form of the (−)strands). The reverse complements of the bisulfite converted methylated(+) stand and (−) stand sequences will be obvious to one of ordinaryskill in the art and are also included by implication in thisdisclosure. Sequences of PCR primers used in amplification of theAmplicons are provided as SEQ ID NOs: 8377-8404 and 8435-8444.

Confirmatory analysis of candidate loci was then done using bisulfitesequencing of candidate loci that were amplified using bisulfitespecific but methylation independent amplification primers. Thisemployed a new sample set of biopsies from: 23 EACs, 8 HGD, 15non-dysplastic BE from individuals without known higher grade lesions.In addition, biopsies were obtained from 5 cases of BE adjacent to HGD,and from 11 cases of BE adjacent to an EAC. These were not included inanalyses of non-dysplastic BE. In addition biopsies were obtained from33 normal squamous mucosa samples.

Table I describes the performance in the confirmatory sample set usingbisulfite sequencing analysis of amplicons of select loci having strongperformance characteristics and identified in the studies discussedabove. In Table 1, columns C-S disclose the performance of the selectamplicons. For each DNA sequence read across each amplicon, the numberof CpGs that were methylated between the amplification primers wascounted, and the read was classified as methylated or unmethyled usingcutoffs for a required number of methylated CpGs on the amplicon. Table1, row 3 lists the number of CpGs between the amplification primers foreach of the amplicons. Table 1, row 4 lists the number of CpGs that needto be methylated on an individual read to count that read as methylated(e.g. for SqBE 2 there are 16 CpG residues between the primers, and14+(meaning >=14) CpGs must be methylated on a read to score it asmethylated. Table 1, rows 6, 7, and 8 record the sensitivity fordetecting esophageal adenocarcinomas (EACs) (row 6), high gradedysplasias (HGD) (row 7) and non-dysplastic Barrett's esophagus(non-dysplastic BE) (row 8), using criteria in which a sample wasdetected if it demonstrated methylation in greater than 10%(0.1) of allDNA reads. Table 1, row 9 records the specificity of each amplicon fornot detecting normal squamous mucosa again using criteria in which asample was detected if it demonstrated methylation in greater than 10%(0.1) of all DNA reads. Table 1, row 11 records the specificity of eachamplicon for not detecting normal squamous mucosa now using criteria inwhich a sample was detected if it demonstrated methylation in greaterthan 1% (0.01) of all DNA reads. As a comparator, Table 1, column Bprovides the same data for detecting methylation in the Vimentin (VIM)locus amplified using primers disclosed in Li et al. (Li M, et al.(2009) Sensitive digital quantification of DNA methylation in clinicalsamples. Nat Biotechnol 27(9):858-863). These primers correspond to SEQID NOs 8445-8446. The amplicon amplified using these primers is derivedfrom the parental (−) strand and is as follows:

Vimentin amplicon (−) strand (SEQ ID NO: 16208):GtTGtttAGGtTGTAGGTGYGGGTGGAYGTAGTtAYGTAGtTtYGGtTGGAGtTYGGtYGGtTYGYGGTGttYGGGtYGtYGAAtATttTGYGGTAGGAG GAYGAGThe reverse complement of SEQ ID NO: 16208 is also generated.

The region of this amplicon lying between the amplification primers, inwhich methylation is analyzed, is as follows:

Vimentin amplicon (−) strand (SEQ ID NO: 16212):GGAYGTAGTtAYGTAGtTtYGGtTGGAGtTYGGtYGGtTYGYGGTGttYG GGtYGtYGAThe reverse complement of SEQ ID NO:16212 is also generated andanalyzed.

For reference, the bisulfite converted sequence that would be derivedfrom amplifying the corresponding bisulfite converted region derivedfrom the Vimentin (+) strand would be.

Vimentin amplicon (+) strand (SEQ ID NO: 16207):tTYGTttTttTAtYGtAGGATGTTYGGYGGttYGGGtAtYGYGAGtYGGtYGAGtTttAGtYGGAGtTAYGTGAtTAYGTttAttYGtAttTAtAGttTG GGtAGtAlong with the reverse complement of SEQ ID NO:16207.And the corresponding portion of SEQ ID NO: 16211 that falls between theprimers used to amplify the Vimentin (−) strand amplicon is:

Vimentin amplicon (+) strand (SEQ ID NO: 16211):TYGGYGGttYGGGtAtYGYGAGtYGGtYGAGtTttAGtYGGAGtTAYGTG AtTAYGTttAlong with reverse complement of SEQ ID NO: 16211.

Amplicons (and patches) need not be used individually, but can becombined into panels for detection of esophageal neoplasia. Examples ofsuch panels, and their associated performance statistics, are providedin Table 1, columns T through AG that provide the markers in the paneland the sensitivity and specificity resulting from the markercombination (when the combination is positive if any member of thecombination is positive).

The sensitivity for detection of EAC (100%), HGD (88%), and BE (100%) isthe same among the combinations shown of: all amplicons, 17 amplicons,15 amplicons, 4 amplicons, three of four combinations of 3 amplicons(columns Y, Z, AA), and for one combination of 2 amplicons (column AF).Specificity for not detecting normal squamous (97%), at a detectioncutoff of 10% of reads being methylated, is the same for allcombinations shown of: 15 amplicons, 4 amplicons, 3 amplicons, or 2amplicons. When specificity is determined using a cutoff of 1% of readsbeing methylated, then among amplicons with the highest sensitivity, thehighest specificity is 94%, demonstrated by the combination of 3amplicons of Table 1, column Z, followed by 91% specificity demonstratedby combinations shown of: 4 amplicons, two combinations of threeamplicons, and one combination of 2 amplicons.

TABLE 1A C D E F G H I J K L M N O P Q R S B SqBE SqBE SqBE SqBE SqBESqBE SqBE SqBE SqBE SqBE SqBE SqBE SqBE SqBE SqBE SqBE SqBE A VIM 2 5 79 10 11-1 11-2 13 14-2 15 16-1 16-2 17-1 18 22-1 22-2 23 Number of  1016  15  27 31 10  17 10 19 22  11 26  26  22 21   9 20 10 CpGs inamplicon analysis Number of   5+ 14+   8+  10+ 16+  5+   8+  5+ 10+ 14+  5+  9+   8+  12+  9+   6+ 11+  6+ CpGs used for methylation callCut-off = 0.1 Sensitivity EAC  70% 74% 100%  40% 65% 96%   4% 87% 38%64%  74% 52%   9%  74% 91%  87% 82% 59% Sensitivity HGD  63% 50% 100% 57% 50% 75%   0% 63% 33% 43%  63% 50%   0%  63% 63%  88% 88% 43%Sensitivity 100% 81%  70%  56% 69% 88%   0% 94% 21% 56% 100% 44%  13%100% 81% 100% 88% 63% for non- dysplasticBarrets Specificity Sq  97% 97%100% 100% 97% 97% 100% 97% 97% 97%  94% 97% 100%  97% 97%  97% 97% 97%Cut-off = 0.01 Specificity Sq  91% 94% 100% 100% 94% 94%  91% 91% 93%91%  91% 94%  94%  94% 94%  94% 94% 94%

TABLE 1B U All 17 SqBE V amplicons: SqBE 15 SqBE 2 SqBE 5 SqBEamplicons: SqBE W 7 SqBE 9 SqBE 5 SqBE 7 SqBE 4 best 10 SqBE 11-1 9 SqBE10 amplicons SqBE 11-2 SqBE 11-1 with highest SqBE 13 SqBE SqBE 11-2 EACcalls 14-2 SqBE 15 SqBE 13 SqBE (SqBE 10 Y T SqBE 16-1 14-2 SqBE 16-1SqBE 11-2 X trio2 Z All SqBE 16-2 SqBE 16-2 SqBE 18 trio1 (SqBE trio3amplicon SqBE 17-1 SqBE 17-1 SqBE 22-1) (SqBE 10 10 SqBE (SqBE 10together, SqBE 18 SqBE SqBE 18 SqBE perform as SqBE 11-2 SqBE 18including 22-1 SqBE 22-2 22-1 SqBE 22-2 well as 16 11-2 SqBE SqBE A VIMSqBE 23 SqBE 23 amplicons SqBE 18) 22-1) 22-1) Number of CpGs inamplicon analysis Number of CpGs used for methylation call Cut-off = 0.1Sensitivity 100% 100% 100% 100% 100% 100% 100% EAC Sensitivity  88%  88% 88%  88%  75%  88%  88% HGD Sensitivity 100% 100% 100% 100% 100% 100%100% for non- dysplastic Barrets Specificity  91%  91%  97%  97%  97% 97%  97% Sq AA trio4 AB AC AD AE AF AG (SqBE double1 double2 doub1e3doub1e4 double5 doub1e6 11-2 (SqBE (SqBE (SqBE (SqBE (SqBE (SqBE SqBE 1810 10 11-2 10 11-2 18 SqBE SqBE SqBE SqBE SqBE SqBE SqBE A 22-1) 11-2)18) 18) 22-1) 22-1) 22-1) Number of CpGs in amplicon analysis Number ofCpGs used for methylation call Cut-off = 0.1 Sensitivity 100% 100% 100%100%  96% 100%  96% EAC Sensitivity  88%  75%  75%  63%  88%  88%  88%HGD Sensitivity 100% 100%  94% 100% 100% 100% 100% for non- dysplasticBarrets Specificity  97%  97%  97%  97%  97%  97%  97% Sq

Confirmatory analysis of candidate loci was further done using bisulfitesequencing of candidate loci that were amplified using the bisulfitespecific but methylation independent amplification primers describedabove. This employed a new sample set of esophageal brushings obtainedusing a cytology brush under endoscopic guidance that were obtainedfirst from 59 control subjects with or without symptomaticgastroesophageal reflux disease (GERD), but all without Barrett'sesophagus (BE). These controls included persons with normal endoscopicfindings or with erosive esophagitis. In these controls brushings wereobtained from the gastroesophageal junction to sample the glandularmucosa, and brushings were also obtained from squamous esophagus mucosa.Brushing were also obtained from esophageal lesions of 107 cases thatincluded 60 individuals with cancers, either adenocarcinoma of theesophagus (EAC) (N=46) or adenocarcinoma of the gastroesophagealjunction (JCA) (N=14), and included 47 individuals with BE. Of BE cases,12 had non-dysplastic short-segment BE (SSBE, <3 cm), 17 hadnondysplastic long segment BE (LSBE ≥3 cm), 8 had low-grade dysplasia(LGD), and 10 had high-grade dysplasia (HGD).

Table 1.5 Columns B-F disclose the performance of individual ampliconsof select loci having strong performance characteristics and identifiedin the studies discussed above in the esophageal brushings sample setanalyzed using bisulfite sequencing performed on a Next Generation DNAsequencing instrument (an Illumina MiSeq instrument). Table 1.5, row 3lists the number of CpGs between the amplification primers for each ofthe amplicons. Table 1.5, row 4, provides the coordinates in hg19 of thegenomic interval that covers the CpGs that lie between the primers (andthat may include several non CpG bases within the primers). Table 1.5,row 5 lists the number of CpGs lying between the primers that wererequired to be methylated on an individual DNA read to count that readas methylated (e.g. for VIM there are 10 CpG residues between theprimers, and 8+(meaning >=8) CpGs were required to be methylated on aread to score the read as methylated). Table 1.5 row 6 lists the minimumpercent of methylated reads (cut-off) identified by bisulfite sequencingthat were required in order to score a sample as methylated and thusdetected (e.g. for VIM >=0.0102 fraction of total reads were required tobe methylated). Table 1.5 rows 7, 8, 9, 10 and 11 record the sensitivityof each amplicon for detecting: esophageal adenocarcinomas (EACs) (row7), adenocarcinomas of the gastroesophageal junction (JCAs) (row 8), lowgrade dysplasias (LGD) (row 9), high grade dysplasias (HGD) (row 10),and non-dysplastic Barrett's esophagus (non-dysplastic BE) (row 11),using criteria in which a sample was detected if it demonstratedmethylation at greater than the cut-off value of row 5. Table 1.5 row 12records the specificity of each amplicon for not detecting normalsquamous esophagus mucosa (normal Sq) and row 13 records the specificityof each amplicon for not detecting normal gastroesophageal junction(GEJ).

In Table 1.5, column B provides data for detecting methylation in theVimentin (VIM) locus amplified using primers disclosed in Li et al. (LiM, et al. (2009) Sensitive digital quantification of DNA methylation inclinical samples. Nat Biotechnol 27(9):858-863). These primers are:

Forward Primer: (SEQ ID 16209) CTCRTCCTCCTACCRCAAAATATTC andReverse Primer: (SEQ ID 16210) GTTGTTTAGGTTGTAGGTGYGGG

(In these notations R denotes alternative sequences that may have eitheran A or G base, and Y denotes alternative sequences that may have eithera C or T base).

The amplicon amplified using these primers is derived from the parental(−) strand and is as follows

Vimentin amplicon (−) strand (SEQ ID NO: 16208):GtTGtttAGGtTGTAGGTGYGGGTGGAYGTAGTtAYGTAGtTtYGGtTGGAGtTYGGtYGGtTYGYGGTGttYGGGtYGtYGAAtATttTGYGGTAGGAG GAYGAGThe reverse complement of SEQ ID NO: 16208 is also generated.The region of this amplicon lying between the amplification primers, inwhich methylation is analysed, is as follows:

Vimentin amplicon (−) strand (SEQ ID NO: 16212):GGAYGTAGTtAYGTAGtTtYGGtTGGAGtTYGGtYGGtTYGYGGTGttYGG GGtYGtYGAThe reverse complement of SEQ ID NO: 16212 is also generated andanalyzed.For reference, the bisulfite converted sequence that would be derivedfrom amplifying the corresponding bisulfite converted region derivedfrom the Vimentin (+) strand would be.

Vimentin amplicon (+) strand (SEQ ID NO: 16207):tTYGTttTttTAtYGtAGGATGTTYGGYGGttYGGGtAtYGYGAGtYGGtYGAGtTttAGtYGGAGtTAYGTGAtTAYGTttAttYGtAttTAtAGttTGGG tAGtAlong with the reverse complement of SEQ ID NO: 16207.And the corresponding portion of SEQ ID NO: 16211 that falls between theprimers used to amplify the Vimentin (−) strand amplicon is:

Vimentin amplicon (+) strand (SEQ ID NO: 16211):TYGGYGGttYGGGtAtYGYGAGtYGGtYGAGtTttAGtYGGAGtTAYGTGA tTAYGTttAlong with reverse complement of SEQ ID NO: 16211.

DNA sequencing reads from each sample were aligned to bisulfiteconverted and unconverted versions of the human reference genome (hg18)using Bowtie2, and the aligned reads were classified as methylated ifthey had 8 or more CpG dinucleotides methylated (out of total of 10 CpGspresent between the primers in the VIM Bisulfite-seq PCR fragment).These analyses were facilitated by the Bismark software, developed forprocessing bisulfite-sequencing data.

A sample was considered methylated for VIM if the methylated vimentinallele frequency was greater than 1.02%, i.e, if more than 1.02% of thesequence reads were classified as methylated (a cut-off that maximizedthe sum of sensitivity plus specificity on the receiver operating curveof all the samples studied). Alternative cutoffs in the range of0.5-3.0% are also possible. The performance of VIM methylation indetecting esophageal lesions is shown in rows 3-12. Overall, at thecutoff of 1.02% methylation, VIM methylation showed sensitivity of 90.7%for identifying BE or esophageal neoplasia and showed specificity forrejecting normal gastroesophageal junction of 93%. When the cutoff forVIM methylation is varied from 0-100%, and sensitivity is plotted versus(100—specificity) (a receiver operating curve), the area under the curvefor the VIM assay=0.949.

In this same sample set, we compared assay of VIM methylation by nextgeneration bisulfite sequencing and the analysis algorithm above (inwhich >=8 methylated CpG is required to score a read as methylatedand >1% of methylated reads defines a cutoff of scoring a sample asmethylated and detected) versus performing assay of VIM methylation byquantitative methylated specific PCR (qMSP) (as described in Moinova etal. Cancer Epidemiol Biomarkers Prev. 2012; 21(4):594-600.). Thebisulfite sequencing method showed superiority with both bettersensitivity and better specificity. In particular, area under thereceiver operating curve for the qMSP assay=0.925, and at the optimalcutoff of 2.2% VIM methylation measured by qMSP (as defined by thereceiver operating curve), assay sensitivity was 82.9% and assayspecificity was 91.3%, which are all inferior to the results obtainedwith the next generation bisulfite sequencing assay and the analysisalgorithm laid out in Table 1.5.

Amplicons need not be used individually, but can be combined into panelsfor detection of esophageal neoplasia. Examples of such panels, andtheir associated performance statistics, are provided in Table 1.5,columns G through L, that specify the markers in the panel and thesensitivity and specificity resulting from the marker combination (whenthe combination is scored positive if any member of the combination ispositive). In these marker combinations, each individual member of thepanel is analysed using the conditions specified for that markerindividually in rows 4 and 5 of columns B-F. The marker combination ofmethylation in amplicons of VIM or of SqBE18 is of particular interest,as the combination of these two markers show detection of 96% ofesophageal adenocarcinomas, 93% of carcinomas of the gastroesophagealjunction, 100% of high grade dysplasias, 100% of low grade dysplasias,and 94% of non-dysplastic long segment Barrett's esophagus, whilepreserving a specificity of 91% for normal gastroesophageal junction.

TABLE 1.5 F F VIM + B C D E VIM + SqBE A VIM SqBE 5 SqBE 16-1 SqBE 18SqBE 5 16-1 Number of CpGs in 10    15    26    21    amplicon analysishg19 Interval covering chr10: chr5: ch11: chr13: CpGs in the amplicon17271466-17271524 1,883,203-1,883,380 110,582,495-110,582,69637,005,877-37,006,009 analysis Number of CpGs used 8+ 13+  15+  16+  formethylation call Cut-off=     0.0105     0.0046     0.0106     0.0312Sensitivity EAC 89%  86%  80%  96%   96%  89% Sensitivity JCA 79%  57% 50%  86%   79%  79% Sensitivity LGD 88%  63%  75%  86%   88%  88%Sensitivity HGD 100%   78%  80%  100%   100% 100% Sensitivity for non-94%  89%  67%  78%  100%  94% dysplastic Barrets Specificity Normal Sq87%  97%  93%  98%   83%  83% Specificity Normal 93%  93%  97%  98%  86%  89% GEJ L I SqBE SqBE J K 5 + F 5 + SqBE SqBE SqBE VIM + SqBE 5 +16-1 + 16-1 + A SqBE 18 16-1 SqBE 18 SqBE 18 SqBE 18 Number of CpGs inamplicon analysis hg19 Interval covering CpGs in the amplicon analysisNumber of CpGs used for methylation call Cut-off= Sensitivity EACSensitivity JCA  96%  90%  98%  96%  98% Sensitivity LGD  93%  64%  86% 93%  93% Sensitivity HGD 100%  75%  86%  86%  86% Sensitivity for non-100%  89% 100% 100% 100% dysplastic Barrets Specificity Normal Sq  94%100% 100%  78% 100% Specificity Normal  87%  93%  95%  92%  92% GEJ  91% 92%  93%  97%  90%

The marker combination of methylation in amplicons of VIM or of SqBE18was further assessed in esophageal cytology brushings ofnormal-appearing gastroesophageal junction (GEJ) or of endoscopicallyvisualized BE or EAC. In these experiments, the same vimentin ampliconsdescribed above were used and contained 10 CpGs. Vimentin specificityfor Normal GEJ samples increases with the requirement that more CpGs inthe read should be unmethylated, in order for the sample to be called“unmethylated.” Conversely, Vimentin Sensitivity for BE/Cancer decreaseswith the requirement that more CpGs in the read are required to becalled “methylated.” The 8+CpG cutoff (blue box in FIG. 2A), maximizesthe sum of specificity for controls and sensitivity for cases. As such,a VIM read was considered methylated if any 8 CpGs out of 10 weremethylated. The output for each sample was the fraction of methylatedreads in the total number of reads for each sample. These values werethen used to generate the Receiver Operating Characteristic (ROC) curve.See FIGS. 1B and 1D that respectively describe training and validationsets of esophageal brushings samples.

The SqBE18 amplicon used on the same samples described in the paragraphabove contained 21 CpGs. These amplicons were generated as describedabove, and were obtained by amplifying bisulfite converted DNA with PCRprimers having the nucleotide sequence of SEQ ID Nos: 8388 and 8402 toderive amplicon sequences having the nucleotide sequence of SEQ ID NO:8318. SqBE18 specificity for Normal GEJ samples increases with therequirement that more CpGs in the read should be unmethylated, in orderfor the sample to be called “unmethylated.” Conversely, SqBE18Sensitivity for BE/Cancer decreases with the requirement that more CpGsin the read are required to be called “methylated.” The 15+, 16+, and17+CpG cutoffs offer identical maximum sensitivity+specificity sum forSqBE18. 16+CpGs (blue box of FIG. 2B), was chosen as the middle of thisrange. As such, a read was considered methylated for the SqBE18 ampliconif any 16 CpGs out of 21 were methylated. The output for each sample wasthe fraction of methylated reads in the total number of reads for eachsample. These values were then used to generate the ROC curve. See FIGS.1A and 1C.

Due to the patchy nature of methylation, normal samples can contain afew random methylated CpGs, while, conversely, the methylated DNA couldcontain some unmethylated CpGs. Different CpG islands have differentmethylation density, and as a result, a cutoff was established for eachregion that would optimally differentiate methylated and unmethylatedDNA. In this study, if greater than 1.05% of VIM reads were methylated,this sample was considered “positive” for VIM methylation, and ifgreater than 3.11% of SqBE18 reads were methylated, this sample wasconsidered “positive” for VIM methylation.

Vimentin and SqBE18 gene methylation (mVIM and mSqBE18) was assayed inDNA samples from either a training set or a validation set of cytologybrushings of the distal esophagus. Both the training and validation setsof brushings of the distal esophagus were from: Unaffected controls(individuals with GERD, erosive esophagitis, or no pathology detectedduring endoscopy—each brushed at the GE Junction); SSBE (short-segmentBarrett's Esophagus (1 to 3 cm)); LSBE (Barrett's Esophagus (3 cm ormore); LGD (Barret's Esophagus with Low-Grade Dysplasia); HGD (Barrett'sEsophagus with High-Grade Dysplasia); Cancer—includes EAC (Esophagealadenocarcinoma) and JCA (Junctional cancer of the esophagus). In thecase of the training sets, samples were scored as methylated when themViM methylated allele frequency was measured as >1.05%, and mSqBE18methylated allele frequency was measured as >3.11% by bisulfitesequencing (representing the ROC cutpoints that provide optimalperformance for each of these assays, respectively). In the case of thevalidation sets, samples were scored as methylated when the mVIMmethylated allele frequency was measured as >1.05%, and mSqBE18methylated allele frequency was measured as >3.11% by bisulfitesequencing (representing the ROC cutpoints from the training set ofbrushings for each of these assays, respectively). For the combinationof the two markers, the calculations were performed in two ways:

-   -   One-marker fails censored: if either mVIM or mSqBE18 sequencing        failed, the sample was excluded from analysis    -   One-marker fails allowed: if one marker failed, the sample was        still included in the analysis and scored as positive or        negative based on the performance of the one working marker.        The data from the training set analyses are summarized in FIG.        3, and the data from the validation set analyses are summarized        in FIG. 4.

All the samples from the training and validation sets of brushingsdescribed in the paragraph above were combined and then scored asmethylated when the mVIM methylated allele frequency was measuredas >1.05%, and mSqBF18 methylated allele frequency was measuredas >3.11% by bisulfite sequencing (representing the ROC cutpoints fromthe training set of brushings for each of these assays, respectively).For the combination of the two markers, the calculations were performedas follows:

-   -   Controls—One-marker fails censored: if either mVIM or mSqBE18        sequencing failed, the sample was excluded from analysis. This        underscores the specificity of the assay.    -   Cases—One-marker fails allowed: if one marker failed, the sample        was still included in the analysis and scored as positive or        negative based on the performance of the one working marker.        This underscores the sensitivity of the assay.        For controls: if one marker failed, the sample was censored in        order not to overestimate the specificity. For cases: if one        marker failed, and the other marker worked, the sample was still        counted. The results of these analyses are provided in FIG. 5.

Esophageal balloon samplings of the distal esophagus were assayed forVIM methylation and ROC curves were obtained. FIG. 6A shows a ROC curvebased on next-generation bisulfite sequencing assay for VIM in thetraining set of 38 controls and 50 cases. Similar to the experimentsdescribed above, the vimentin amplicon that was used contained 10 CpGs,and a read was considered methylated, if any 8 CpGs out of 10 aremethylated (based on the analysis of data in slide 31. The output foreach sample was the fraction of methylated reads in the total number ofreads for each sample. These values were then used to generate the ROCcurve Optimal ROC cutpoint for mVIM assay on balloons was between 0.95%and 1.16%, and 1% was chosen as a convenient number in the middle ofthis range. The actual cutoff picked by MedCalc for VIM balloons was0.95%.

Esophageal balloon samplings of the distal esophagus were also assayedfor SqBE18 methylation and ROC curves were obtained. FIG. 6B shows a ROCcurve based on next-generation bisulfite sequencing SqBE18 assay in thetraining set of 38 controls and 50 cases. Similar to the experimentsdescribed above, the SqBE18 amplicon that was used contained 21 CpGs,and a read was considered methylated if any 16 CpGs out of 21 weremethylated. The output for each sample was the fraction of methylatedreads in the total number of reads for each sample. These values werethen used to generate the ROC curve. Optimal ROC cutpoint for SqBE18assay on balloons was between 0.1%, based on maximizing the sum ofsensitivity and specificity. However, a higher cutpoint was chosen tomaintain higher specificity of the assay. Any cutpoint between 0.76% and1.06%, would give the same value of sensitivity and specificity. 1% waschosen as the convenient number in the middle of this range. The actualcutoff picked by MedCalc for SqBE18 balloons was 0.1%. However, 1% waschosen as the cutoff to maximize specificity. Any number between 0.76%and 1.06% would have worked the same as 1%. If >0.1%, then thesensitivity: 84.0%, and the specificity 94.7%.

Vimentin and SqBE18 gene methylation (mVIM and mSqBE18) was assayed inesophageal balloon DNA samples of the distal esophagus from: Unaffectedcontrols (individuals with GERD, erosive esophagitis, or no pathologydetected during endoscopy; SSBE (short-segment Barrett's Esophagus (1 cmto less than 3 cm)); LSIBE (Barrett's Esophagus (3 cm or more); LGD(Barret's Esophagus with Low-Grade Dysplasia); HGD (Barrett's Esophaguswith High-Grade Dysplasia); Cancer—includes EAC (Esophagealadenosarcoma) and JCA (Junctional cancer of the esophagus). Samples werescored as methylated when the mVIM and SqBE18 methylated allelefrequency was measured as >1%, based on balloon ROC curves described inthe preceding two paragraphs. The results from these analyses areprovided in FIG. 7.

Example 2: Identification of Esophageal Cancer Informative Loci toDetect Progression of Esophageal Neoplasia

Discovery data were also analyzed for each individual CpG residue in theRRBS data set to identify loci that could be used to distinguish EACfrom BE. Individual CpGs were considered methylated in EAC versus BE ifthey showed methylation of less than 10% of reads of all informative BEsamples, where at least 3 BE samples were informative, and if theyshowed methylation of less than 10% of reads of all informative normalsquamous samples, and where an informative sample had equal to orgreater than 20 reads covering the CpG, and if 6 or more of the EACsamples demonstrated percent methylation at a level that was at least 20percentage points greater than the methylation level of the mostmethylated BE sample. CpGs meeting criteria for methylation in EACversus and BE are defined as methylated in EAC vs BE. Such methylatedCpGs were then aggregated into patches in instances in which methylatedCpGs were within 200 bp one another.

186 genomic patches defined as methylated in EACs versus BE in thediscovery set were identified (see SEQ ID NOs: 8447-8818). The genomiccoordinates of the genomic patches defined as methylated by the abovecriteria were also recorded. The genomic sequences of these patches onthe respective genomic (+) and (−) strands were determined and recorded.(Upper and lower case designations were used according to those of theUCSC browser, where lower case sequences are lower complexity DNAsequences). The bisulfite converted sequences of these correspondingpatches (i.e, the bisulfite converted sequence of the (+) strand and thebisulfite converted sequence of the (−) strand) were determined andrecorded (see sequences of SEQ ID NOs: 8819-9004 and 9191-9376 for thebisulfite converted sequences of the respective (+) and (−) strands). Cresidues that may be methylated or unmethylated, and hence may bebisulfite converted to T (if unmethylated) or remain as a C (ifmethylated), were designated with a Y (where Y denotes C or T), andwhere, after bisulfite conversion, actual maintenance of a Y designatedbase as a C was scored as methylation at that base. Thus, thesesequences represent the group of all combinations of all sequences inwhich 0, 1, or more than one Y is converted to a T. The reversecomplements of the bisulfite converted sequences of the (+) and (−)strands will be obvious to one of ordinary skill in the art and are alsoincluded by implication in this disclosure. The bisulfite convertedsequences of the fully methylated form of the corresponding patches(i.e, in which all Y bases in every bisulfite converted sequence areretained as a C), corresponding to the (+) strand the (−) strand weredetermined and recorded (see sequences of SEQ ID NOs: 9005-9190 and9377-9562 for the bisulfite converted sequences of the fully methylatedform of the (+) and (−) strands respectively of the correspondingpatches). The reverse complements of the bisulfite converted methylated(+) stand and (−) stand sequences will be obvious to one of ordinaryskill in the art and are also included by implication in thisdisclosure.

Patches were expanded by 100 base pairs on either side so as toaccommodate either the design of amplification primers or to excludeadditional presumptively methylated bases. The sequences of theseexpanded patches correspond to SEQ ID NOs: 9563-9934 and their genomiccoordinates were also recorded. The genomic sequences of these expandedpatches on the respective genomic (+) and (−) strands were determinedand recorded. (Upper and lower case designations were used according tothose of the UCSC browser, where lower case sequences are lowercomplexity DNA sequences). The bisulfite converted sequences of thesecorresponding expanded patches (i.e, the bisulfite converted sequence ofthe (+) strand and the bisulfite converted sequence of the (−) strand)were determined and recorded (see sequences of SEQ ID NOs: 9935-10120and 10307-10492 for the bisulfite converted sequences of the respective(+) and (−) strands). C residues that may be methylated or unmethylated,and hence may be bisulfite converted to T (if unmethylated) or remain asa C (if methylated), were designated with a Y (where Y denotes C or T),and where, after bisulfite conversion, actual maintenance of a Ydesignated base as a C was scored as methylation at that base. Thus,these sequences represent the group of all combinations of all sequencesin which 0, 1, or more than one Y is converted to a T. The reversecomplements of the bisulfite converted sequences of the (+) and (−)strands will be obvious to one of ordinary skill in the art and are alsoincluded by implication in this disclosure. The bisulfite convertedsequences of the fully methylated form of the corresponding expandedpatches (i.e, in which all Y bases in every bisulfite converted sequenceare retained as a C), corresponding to the (+) strand the (−) strandwere determined and recorded (see sequences of SEQ ID NOs: 10121-10306and 10493-10678 for the bisulfite converted sequences of the fullymethylated form of the (+) and (−) strands respectively of thecorresponding expanded patches). The reverse complements of thebisulfite converted methylated (+) stand and (−) stand sequences will beobvious to one of ordinary skill in the art and are also included byimplication in this disclosure.

CpG islands overlapping patches that may contain additional CpGs thatare methylated coordinately with patches were also defined. Thesequences of these CpG islands correspond to SEQ ID NOs: 10679-10972.The genomic coordinates of the CpG islands were also recorded. Thegenomic sequences of these expanded patches on the respective genomic(+) and (−) strands were determined and recorded. (Upper and lower casedesignations were used according to those of the UCSC browser, wherelower case sequences are lower complexity DNA sequences). The bisulfiteconverted sequences of these corresponding CpG islands (i.e, thebisulfite convened sequence of the (+) strand and the bisulfiteconverted sequence of the (−) strand) were determined and recorded (seesequences of SEQ ID NOs: 10973-11119 and 11267-11413 for the bisulfiteconverted sequences of the respective (+) and (−) strands) C residuesthat may be methylated or unmethylated, and hence may be bisulfiteconverted to T (if unmethylated) or remain as a C (if methylated), weredesignated with a Y (where Y denotes C or T), and where, after bisulfiteconversion, actual maintenance of a Y designated base as a C was scoredas methylation at that base. Thus, these sequences represent the groupof all combinations of all sequences in which 0, 1, or more than one Yis converted to a T. The reverse complements of the bisulfite convertedsequences of the (+) and (−) strands will be obvious to one of ordinaryskill in the art and are also included by implication in thisdisclosure. The bisulfite converted sequences of the fully methylatedform of the corresponding CpG islands (i.e, in which all Y bases inevery bisulfite converted sequence are retained as a C), correspondingto the (+) strand the (−) strand were determined and recorded (seesequences of SEQ ID NOs: 11120-11266 and 11414-11266 for the bisulfiteconverted sequences of the fully methylated form of the (+) and (−)strands respectively of the corresponding CpG islands). The reversecomplements of the bisulfite converted methylated (+) stand and (−)stand sequences will be obvious to one of ordinary skill in the art andare also included by implication in this disclosure.

Regions of Interest (ROI) were defined that provided preferred regionsfor design of PCR amplicons that would encompass preferred patches. Thegenomic coordinates of the ROI were also recorded. The sequences of the(+) strands of the Regions of Interest correspond to SEQ ID NOs:12563-12568, and the sequences of the (−) strands Regions of Interestcorrespond to SEQ ID NOs: 12581-12586. (Upper and lower casedesignations were used according to those of the UCSC browser, wherelower case sequences are lower complexity DNA sequences). The bisulfiteconverted sequences of these corresponding Regions of Interest (i.e, thebisulfite converted sequence of the (+) strand and the bisulfiteconverted sequence of the (−) strand) were determined and recorded (seesequences of SEQ ID NOs: 12569-12574 and 12587-12592 for the bisulfiteconverted sequences of the respective (+) and (−) strands). C residuesthat may be methylated or unmethylated, and hence may be bisulfiteconverted to T (if unmethylated) or remain as a C (if methylated), weredesignated with a Y (where Y denotes C or T), and where, after bisulfiteconversion, actual maintenance of a Y designated base as a C was scoredas methylation at that base. Thus, these sequences represent the groupof all combinations of all sequences in which 0, 1, or more than one Yis converted to a T. The reverse complements of the bisulfite convertedsequences of the (+) and (−) strands will be obvious to one of ordinaryskill in the art and are also included by implication in thisdisclosure. The bisulfite converted sequences of the fully methylatedform of the Regions of Interest (i.e in which all Y bases in everybisulfite converted sequence are retained as a C), corresponding to the(+) strand the (−) strand were determined and recorded (see sequences ofSEQ ID NOs: 12575-12580 and 12593-12598 for the bisulfite convertedsequences of the fully methylated form of the (+) and (−) strandsrespectively of the corresponding Regions of Interest). The reversecomplements of the bisulfite converted methylated (+) stand and (−)stand sequences will be obvious to one of ordinary skill in the art andare also included by implication in this disclosure.

Specific PCR Amplicons were defined within the Regions of Interest(ROI). The genomic coordinates of the Amplicons were recorded. Thesequences of the (+) strands of the Amplicons correspond to SEQ ID NOs:12599-12604 and 12647-12649, and the sequences of the (−) strands of theAmplicons correspond to SEQ ID NOs: 12617-12622 and 12656-12658. (Upperand lower case designations were used according to those of the UCSCbrowser, where lower case sequences are lower complexity DNA sequences).The bisulfite converted sequences of these Amplicons (i.e, the bisulfiteconverted sequence of the (+) strand and the bisulfite convertedsequence of the (−) strand) were determined and recorded (see sequencesof SEQ ID NOs: 12605-12610 and 12650-12652 for the bisulfite convertedsequences of the (+) strands and see sequences of SEQ ID NOs:12623-12628 and 12659-12661 and for the bisulfite converted sequences ofthe (−) strands). C residues that may be methylated or unmethylated, andhence may be bisulfite converted to T (if unmethylated) or remain as a C(if methylated), were designated with a Y (where Y denotes C or T), andwhere, after bisulfite conversion, actual maintenance of a Y designatedbase as a C was scored as methylation at that base. Thus, thesesequences represent the group of all combinations of all sequences inwhich 0, 1, or more than one Y is converted to a T. The reversecomplements of the bisulfite converted sequences of the (+) and (−)strands will be obvious to one of ordinary skill in the art and are alsoincluded by implication in this disclosure. The bisulfite convertedsequences of the fully methylated form of the Amplicons (i.e, in whichall Y bases in every bisulfite converted sequence are retained as a C),corresponding to the (+) strand the (−) strand were determined andrecorded (see sequences of SEQ ID NOs: 12611-12616 and 12653-12655 forthe bisulfite converted sequences of the fully methylated form of the(+) strands and see sequences of SEQ ID NOs: 12629-12634 and 12662-12664for the bisulfite converted sequences of the fully methylated form ofthe (−) strands). The reverse complements of the bisulfite convertedmethylated (+) stand and (−) stand sequences will be obvious to one ofordinary skill in the art and are also included by implication in thisdisclosure. Sequences of PCR primers used in amplification of theAmplicons are provided as SEQ ID NOs. 12635-12646 and 12665-12670.

Confirmatory analysis of candidate loci was then done using bisulfitesequencing of candidate loci that were amplified using bisulfitespecific but methylation independent amplification primers. Thisemployed a new sample set of biopsies from: 23 EACs, 8 HGD, 15non-dysplastic BE from individuals without known higher grade lesions.In addition, biopsies were obtained from 5 cases of BE adjacent to HGD,and from 11 cases of BE adjacent to an EAC. These are not included inanalyses of non-dysplastic BE. In addition biopsies were obtained from33 normal squamous mucosa samples.

Table 2 describes the performance in the confirmatory sample set usingbisulfite sequencing analysis of amplicons from select loci defined asmethylated in EACs versus BE and having preferred markercharacteristics. In Table 2, Columns B-J disclose the performance of theamplicons in the confirmatory data set. In this data set, methylationwas calculated as the average level of methylation of all CpGs inbetween the primers for amplifying the amplicon. For each read acrosseach amplicon the number of CpGs that were methylated was counted andthe read was classified as methylated or unmethyled using cutoffs for arequired number of methylated CpGs on the amplicon. Table 2, row 3 liststhe number of CpGs between the amplification primers for each of theamplicons. Table 2, row 4 lists the number of CpGs that need to bemethylated on an individual read to count that read as methylated (e.g.for Up3 there are 36 CpG residues between the primers, and25+(meaning >=25) CpGs must be methylated on a read to score it asmethylated). Table 2, rows 6 and 7 record the sensitivity for detectingEACs (row 6) and HGD (row 7) using criteria in which a sample wasdetected if it demonstrated methylation in greater than 10% (0.1) of allDNA reads Table 2, rows 8 and 9 record the specificity of each ampliconfor not detecting non-dysplastic BE (row 8) and for not detecting normalsquamous mucosa (row 9) again using criteria in which a sample wasdetected if it demonstrated methylation in greater than 10% (0.1) of allDNA reads. Table 2, rows 11 and 12 record the specificity of eachamplicon for not detecting non-dysplastic BE (row 11) and for notdetecting normal squamous mucosa (row 12) using criteria in which asample was detected if it demonstrated methylation in greater than 1%(0.01) of all DNA reads. Amplicons (and patches) need not be usedindividually, but can be combined into panels for detection ofesophageal neoplasia. Performance statistics of selected panels ofamplicons are provided in Table 2 columns K through V that provides thesensitivity and specificity of the panels (when the combination ispositive if any member of the combination is positive).

TABLE 2A B C D E F G H I J A Up3 Up10 Up15_ampl1 Up15_ampl2 Up20_ampl1Up20_ampl2 Up27 Up35_ampl1 Up35_ampl2 Number of CpGs in amplicon  36  30 25  26  21  16  21  26  18 analysis Number of CpGs used for  25+  17+ 15+ 15+  11+   6+  11+  14+  10+ methylation call Cut-off = 0.1Sensitivity EAC  15%  40%  22% 26%  37%  0%  20%  45%  30% SensitivityHGD   0%  25%  13%  0%  33%  0%  17%  17%  25% Specificity fornon-dysplastic BE 100% 100% 100% 94% 100% 100% 100% 100% 100%Specificity Sq 100% 100% 100% 97% 100% 100%  97% 100% 100% Cut-off =0.01 Specificity for non-dysplastic BE 100% 100% 100% 94%  94% 100% 100%100% 100% Specificity Sq 100%  88% 100% 91%  90% 100%  93% 100% 100%

TABLE 2B K All ampli- cons M N together: L 7 7 Up3, 6 core ampliconamplicon Up10, ampli- combo 1: combo 2: R S T U V Up15-1, con: up10 andUp35-2 O P Q 4 4 4 4 4 Up15-2, Up3, Up3, and Up3, 2 3 3 ampliconamplicon amplicon amplicon amplicon Up20-1, Up15-1, Up15-1, Up15-1,ampli- mplicon amplicon combo 1: combo 2: combo 3: combo 4: combo 5:Up20-2, Up15-2, Up15-2, Up15-2, con combo 1 combo 2: Up15-1, Up15-1,Up15-1, Up15-1, Up15-1, Up27, Up20-1, Up20-1, Up20-1, combo: Up15-1,Up15-1, Up35-1, Up35-1, Up35-1, Up35-1, Up35-1, Up35-1, Up27, Up27,Up27, Up15-1, Up35-1, Up35-1, Up10, Up10, Up15-2, Up15-2, Up20-1, AUp35-2 Up35-1 Up35-1 U035-1 Up35-1 Up10 Up20-1 Up15-2 Up27 Up20-1 Up27Up27 Number of CpGs in amplicon analysis Number of CpGs used formethylation call Cut-off = 0.1 Sensitivity 65% 65% 65% 65%  52%  57% 57% 61%  61% 61% 61%  61% EAC Sensitivity 50% 38% 50% 50%  25%  38% 38% 38%  38% 38% 25%  38% HGD Specificity for 94% 94% 94% 94% 100% 100%100% 94% 100% 94% 94% 100% non-dysplastic BE Specificity Sq 94% 94% 94%94% 100% 100% 100% 97%  97% 97% 94%  97% Cut-off = 0.01 Specificity for88% 88% 88% 88% 100% 100%  94% 94% 100% 88% 94%  94% non-dysplastic BESpecificity Sq 82% 82% 82% 82% 100%  94%  91% 88%  91% 85% 88%  88%

In addition, RRBS discovery data was analyzed to identify CpG residuesthat demonstrated: i) at least 3 informative BE, which in everyinformative BE demonstrated at least 90% methylation; and where ii) nomore than 5% of informative normal squamous samples demonstratedmethylation level below 90%, and that iii) demonstrated at least 6informative EAC, where in these informative EACs the level ofmethylation was at least 20 percentage points lower than the methylationlevel of the least methylated BE. CpGs meeting these criteria aredefined as unmethylated in EAC versus BE. Such unmethylated CpGs werethen aggregated into patches by grouping together unmethylated CpGs thatwere within 200 bp of one another. Unmethylated in EAC patches mayconsist of 1 CpG up to any number of CpGs that meet the above criteria.

Example 3: Identification of Esophageal Cancer Informative Loci toDetect Progression of Esophageal Neoplasia

Biopsy samples (that overlapped with the confirmatory biopsy sample set)were further analyzed in tests of panels of markers for detecting theprogression of Barrett's esophagus to Barrett's esophagus high gradedysplasia (HGD) or to esophageal adenocarcinoma (EAC). Three panels ofmarkers were selected for study. The first marker panel consisted ofdetecting at least one of the following four methylated markers: Up15-1,Up35-1, Up27, and Up10 (using bisulfite sequencing analysis of thecorresponding amplicons and using the criteria for detection specifiedin table 2A). The second panel consisted of testing for somaticnon-synonymous mutations in TP53 in assays in which TP53 was amplifiedfrom genomic DNA using a set of PCR amplicons that spanned the TP53coding region and in which Next Generation DNA Sequencing was then usedto compare TP53 sequences from esophageal lesions versus matched normalesophagus tissue. Samples were classified as detected if a TP53 mutantallele frequency of greater than or equal to 10% was identified. Thethird panel was a combination of detection of methylation in any ofUp15-1, Up35-1, Up27, and Up10 or detection of mutation in TP53.

Table 3 shows the individual performance of biomarkers in detecting thedifferent sample types of the 1st validation biopsies set usingdifferent cut-off criteria for methylation than the analysis of table 2Shown is the performance for detection of different sample types of 5different methylated DNA markers tested by bisulfite sequencing analysisof the corresponding amplicons (Up3, Up10, Up27, Up35-1, Up35-2). Table3 specifies the number of CpGs required to be methylated on a DNAsequence read to classify that read as methylated for this analysis.Results are presented when samples are considered methylated if greaterthan or equal to 1% of all DNA sequence reads are classified asmethylated, or if greater than or equal to 10% of all DNA sequence readsare classified as methylated. Also shown is the performance fordetecting samples of testing for non-synonymous somatic mutation inassays in which TP53 was amplified from genomic DNA using a set of PCRamplicons that spanned the TP53 coding region and in which NextGeneration DNA Sequencing was then used to compare TP53 sequences fromesophageal lesions versus matched normal esophagus tissue. Shown arerates of sample detection (expressed as sensitivity or specificity) whensamples are classified as TP53 mutant if greater than or equal to 3% ofTP53 reads are scored as mutant, or if greater than or equal to 10% ofTP53 reads are scored as mutant.

TABLE 3 Unchanged CpG # cut-off applied to all Updated CpG # Cut-offsample sets applied to all sample sets Up3 Up35-1 Up35-2 Up10 Up27 25 +CpGs 14 + CpGs 10 + CpGs 26 + CpGs 15 + CpGs p53 Category >1% =positive >1% = positive >1% = positive >1% = positive >1% = positive >3%Sensitivity  20%  50%  35%   30%  20%  70% EAC Sensitivity   7%  20% 19%   0%   7%  25% or BEs synchronous to EAC or HGD Sensitivity  17% 33%  38%   0%  17%  25% HGD Specificity 100% 100% 100% 100%  97% 100%Sq Specificity 100% 100% 100% 100% 100% 100% for non- dysplastic BE >10%= positive >10% = positive >10% = positive >10% = positive >10% =positive >10% = positive Sensitivity  15%  45%  30%  10%  15%  61% EACSensitivity   7%   7%   6%   0%   7%  19% for BEs matching higherpathologies, either EAC or HGD Sensitivity   0%  17%  25%   0%  17%  25%HGD

Table 4 shows the performance of selected combinations of the methylatedDNA markers (Up3, Up10, Up27, Up35-1, Up35-2) for detection of differentsample types in the experiment presented in Table 3. Samples are scoredas methylated if any member of the marker combination panel scores thesample as methylated. Results are presented when the individual markersare considered methylated if greater than or equal to 1% of all DNAsequence reads are classified as methylated, or if greater than or equalto 10% of all DNA sequence reads are classified as methylated.

TABLE 4A Cut-off for positivity (e.i. samples is positive if more thanthis % of reads are methylated at the given Up35-1 Up35-1 Up35-1 Up35-2Up35-2 Up10 Up3. Up3. number of Up3 and Up3 and Up3 and Up3 and and andand and and and Up35-1, Up35-1, CpGs) Category Up35-1 Up35-2 Up10 Up27Up35-2 Up10 Up27 Up10 Up27 Up27 Up35-2 Up10  1% Sensitivity  50%  45% 50%  35%  50%  60%  60%  50%  50%  40%  50%  60% EAC  1% Sensitivity 20%  20%  11%  13%  27%  11%  20%  11%  27%   0%  27%  11% for BEssynchronous to EAC or HGD  1% Sensitivity  33%  33%  25%  33%  33%  50% 50%  50%  50%   0%  33%  50% HGD  1% Specificity Sq 100% 100% 100%  97%100% 100%  97% 100%  97%  94% 100% 100%  1% Specificity 100% 100% 100%100% 100% 100% 100% 100% 100% 100% 100% 100% for non- dysplastic BE 10%Sensitivity  45%  35%  30%  25%  45%  50%  50%  30%  40%  20%  45%  50%EAC Sensitivity  13%  13%  11%  13%   7%   0%  13%   0%  13%   0%  13% 11% for BEs matching higher pathologies, either EAC or HGD 10%Sensitivity  17%  33%   0%  17%  33%  25%  33%  50%  50%   0%  33%  25%HGD

TABLE 4B Cut-off for positivity (e.i. samples is positive if more thanthis % of reads are methylated Up3. Up3. Up35-1, Up3, Up3, at the givenUp3. Up3. Up3. Up3. Up35-1, Up35-2, Up35-1, Up35-1, Up35-2, Up35-2,Up35-1, All 5 number of Up35-1, Up35-2, Up35-2, Up10, Up10, Up10,Up35-2, Up35-2, Up10 Up10 Up10 markers CpGs) Category Up27 Up10 Up27Up27 up27 up27 Up10 Up27 Up27 Up27 Up27 together  1% Sensitivity EAC 60%  60%  55%  60%  70%  60%  60%  60%  70%  70%  70%  70%  1%Sensitivity for BEs  20%  11%  27%  11%  11%  11%  11%  27%  11%  11% 11%  11% synchronous to EAC or HGD  1% Sensitivity HGD  50%  50%  50% 25%  50%  50%  50%  50%  50%  50%  50%  50%  1% Specificity Sq  97%100%  97%  94%  94%  94% 100%  97%  94%  94%  94%  94%  1% Specificityfor 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100%non-dysplastic BE 10% Sensitivity EAC  50%  30%  40%  40%  60%  40%  50% 50%  40%  40%  60%  60% Sensitivity for BEs  20%  11%  20%  11%   0%  0%  11%  20%  11%  11%  11%  11% matching higher pathologies, eitherEAC or HGD 10% Sensitivity HGD  33%  50%  50%   0%  25%  50%  50%  50% 50%  50%  25%  50%

Samples summarized in Table 3 and Table 4 were additionally tested fornon-synonymous somatic mutations in TP53. Table 5 shows performance ofselected combinations of methylated DNA markers (Up3, Up10, Up27,Up35-1, Up35-2) plus testing for mutations in TP53 (p53) for detectionof different sample types. Samples are scored as detected if any memberof the marker combination panel scores the sample as methylated or ifanalysis for TP53 mutations scores the sample as TP53 mutant. Shown isthe performance of the marker panel in which samples are scored asdetected if any methylation marker is detected as methylated at greaterthan or equal to 1% of DNA reads, or if TP53 is detected as mutant atgreater than or equal to 10% of the DNA sequence reads. Also shown isthe performance of the marker panel in which samples are scored asdetected if any methylation marker is detected as methylated at greaterthan or equal to 10% of DNA reads, or if TP53 is detected as mutant atgreater than or equal to 10% of the DNA sequence reads.

TABLE 5A Cut-off for positivity (e.i. sample is positive if more thanthis % of reads are methylated at the given Up-3 and Up35-1 and Up35-2and Up10 and number of CpGs) P53 cut-off Category p53 p53 p53 p53 Up27and p53  1% 10% Sensitivity EAC  70%  80%  70%  60%  70%  1% 10%Sensitivity for BEs  20%  20%  19%  11%  27% synchronous to EAC or HGD10% Sensitivity HGD  33%  50%  38%  25%  33%  1% 10% Specificity Sq 100%100% 100% 100%  97%  1% 10% Specificity for non- 100% 100% 100% 100%100% dysplastic BE Individual Methylation Markers + P53 10% 10%Sensitivity EAC  70%  80%  70%  60%  70% 10% Sensitivity for BEs  20% 20%  19%  11%  27% matching higher pathologies, either EAC or HGD 10%10% Sensitivity HGD  33%  50%  38%  25%  33%

TABLE 5B Cut-off for positivity (e.i. sample is positive if more thanthis % of reads are methylated Up35-1, Up3, at the Up3 Up3 Up35-2 Up35-2Up10 Up3. Up3. Up3. Up35-2, Up35-2, Up35-2, given Up3 and and and andand and Up35-2, Up35-2, Up10, Up10, Up10 Up10 number P53 Up35-2 Up10Up27 Up10 Up27 Up27 Up10 Up27 Up27 up27 Up27 Up27 of CpGs) cut-offCategory and p53 and p53 and p53 and p53 and p53 and p53 and p53 and p53and p53 and p53 and p53 and p53  1% 10% Sensitivity  80%  80%  80%  70% 80%  80%  60%  85%  90%  80%  90%  90% EAC  1% 10% Sensitivity  20% 11%  27%  11%  27%  11%  11%  27%  11%  11%  11%  11% for BEs synchro-nous to EAC or HGD 10% Sensitivity  50%  25%  33%  50%  50%  25%  50% 50%  25%  50%  50%  50% HGD  1% 10% Specificity 100% 100%  97% 100% 97%  94% 100%  97%  94%  94%  94%  94% Sq  1% 10% Specificity 100% 100%100% 100% 100% 100% 100% 100% 100% 100% 100% 100% for non- dysplastic BEMethylation Marker Combinations Merged with P53 Mutation 10% 10%Sensitivity  75%  70%  75%  70%  80%  70%  30%  80%  80%  80%  80%  80%EAC 10% Sensitivity  20%  11%  27%  11%  27%  11%  11%  27%  11%  11% 11%  11% for BEs matching higher pathologies, either EAC or HGD 10% 10%Sensitivity  50%  25%  33%  50%  50%  25%  50%  50%  25%  50%  50%  50%HGD

DNA was also extracted from esophageal samples that were also obtainedby cytology brushings of the esophagus. The sample set includedbrushings from 49 esophageal adenocarcinomas (EAC); 14 carcinomas of thegastroesophageal junction (JCA); 8 Barrett's esophagus with low gradedysplasia (LGD); 9 Barrett's esophagus with high-grade dysplasia (HGD);33 cases of Barrett's esophagus without dysplasia from cases without HGDor EAC, otherwise termed non-dysplastic BE, that included 13 cases ofshort segment Barrett's esophagus (SSBE). Also included were brushingsof the gastroesophageal junction (normal GEJ) from 62 individualswithout Barrett's esophagus, without HGD, without EAC. This includedindividuals with gastroesophageal reflux disease, with eosinophilicesophagitis, or without any disease. Also included were 176 brushings ofnormal squamous esophagus from each of the above individuals. These DNAsamples were analyzed for methylation by bisulfite sequencing ofselected amplicons and were also analyzed for non-synonymous somaticmutations in TP53.

Table 6 shows the individual performance of biomarkers in detecting thedifferent sample types of the validation brushings set. Shown is theperformance for detection of different sample types of 5 differentmethylated DNA markers analyzed by bisulfite sequencing of selectedamplicons (Up3, Up10, Up27, Up35-1, Up35-2). The table specifies thenumber of CpGs required to be methylated on a DNA sequence read toclassify that read as methylated. Results are presented when samples areconsidered methylated if greater than or equal to 1% of all DNA sequencereads are classified as methylated, or if greater than or equal to 10%of all DNA sequence reads are classified as methylated. Also shown isthe performance for detecting samples of testing for somatic mutation inassays in which TP53 was amplified from genomic DNA using a set of PCRamplicons that spanned the TP53 coding region and in which NextGeneration DNA Sequencing was then used to compare TP53 sequences fromesophageal lesions versus matched normal esophagus tissue. Shown arerates of sample detection (expressed as sensitivity or specificity) whensamples are classified as TP53 mutant if greater than or equal to 3% ofTP53 reads are scored as mutant, or if greater than or equal to 10% ofTP53 reads are scored as mutant.

TABLE 6A Unchanged CpG # cut-off applied to all sample sets Up-3 Up35-1Up35-2 Category 25 + CpGs 14 + CpGs 10 + CpGs >1% = positive >1% =positive >1% = positive Sensitivity EAC 44% 57% 57% Sensitivity JCA 14%43% 21% Sensitivity LGD 25% 38% 25% Sensitivity HGD 67% 56% 50%Specificity normal GEJ 98% 98% 98% Specificity for “non-dysplastic 95%100%  100%  BE”- excluding SSBE Specificity for all BE without 97% 85%94% dysplasia (including SSBE and non-dysplastic BE >10% = positive >10%= positive >10% = positive Sensitivity EAC 21% 57% 45% Sensitivity JCA 7% 29%  7% Sensitivity LGD 13% 38% 25% Sensitivity HGD 33% 56% 100% Specificity normal GEJ 100%  100%  100%  Specificity for “non-dysplastic100%  100%  100%  BE”- excluding SSBE Specificity for all BE without100%  88% 97% dysplasia (including SSBE and non-dysplastic BE)

TABLE 6B Updated CpG # Cut-off applied to all sample sets Up10 Up27Category 26 + CpGs 15 + CpGs p53 >1% = positive >1% = positive >3%Sensitivity EAC 31% 43% 59% Sensitivity JCA 43% 14% 50% Sensitivity LGD 0% 13% 13% Sensitivity HGD 22% 20% 40% Specificity normal GEJ 98% 100% 100%  Specificity for “non-dysplastic 100%  95% 100%  BE”- excludingSSBE Specificity for all BE without 100%  94% 97% dysplasia (includingSSBE and non-dysplastic BE >10% = positive >10% = positive >10% =positive Sensitivity EAC 25% 29% 47% Sensitivity JCA 29%  7% 29%Sensitivity LGD  00% 13%  0% Sensitivity HGD 11% 20% 40% Specificitynormal GEJ 100%  100%  100%  Specificity for “non-dysplastic 100'%  95%100%  BE”-excluding SSBE Specificity for all BE without 100'%  97% 97%dysplasia (including SSBE and non-dysplastic BE)

Table 7 shows the performance of selected combinations of the methylatedDNA markers (Up3, Up10, Up27, Up35-1, Up35-2) for detection of differentsample types in the esophageal brushings samples presented in Table 6.Samples are scored as methylated if any member of the marker combinationpanel scores the sample as methylated. Results are presented when theindividual markers are considered methylated if greater than or equal to1% of all DNA sequence reads are classified as methylated, or if greaterthan or equal to 10% of all DNA sequence reads are classified asmethylated.

TABLE 7A Cut-off for positivity (e.i. sample is positive if more thanthis % of reads are methylated Up3 Up3 Up3 Up3 Up35-1 Up35-1 Up35-1Up35-2 Up35-2 Up10 Up3

at the given and and and and and and and and and and Up35-1, number ofCpGs) Category Up35-1 Up35-2 Up10 Up27 Up35-2 Up10 Up27 Up10 Up27 Up27Up35-2  1% Sensitivity EAC 63% 60% 57% 56%  61%  71% 67%  71% 63% 5

% 63%  1% Sensitivity JCA 43% 21% 50% 29%  43%  57% 50%  50% 36% 43% 4

%  1% Sensitivity LGD 38% 33% 1

% 25%  33%  29% 38%  29% 25% 14% 38%  1% Sensitivity HGD 78% 78% 78% 67% 56%  78% 67%  67% 60% 33% 78%  1% Specificity normal GEJ 98% 98% 97%98%  98%  97% 9

%  97% 98% 9

% 98%  1% Specificity for 95% 95% 95% 90% 100% 100% 95% 100% 95% 95% 95%non-dysol BE  1% Specificity all BE 84% 94% 97% 91%  85%  85% 82%  94%91% 94% 84% without dysplasia(inclu- ding SSBE, and non-dysplastic BE)10% Sensitivity EAC 56% 48% 38% 38%  57%  67% 65%  58% 55% 42% 63% 10%Sensitivity JCA 29%  7% 29% 14%  29%  43% 36%  29% 14% 36% 29% 10%Sensitivity LGD 38% 38%  0% 25%  38%  29% 38%  29% 25% 14% 38% 10%Sensitivity HGD 67% 44% 33% 44%  56%  67% 67%  44% 50% 22% 78%

indicates data missing or illegible when filed

TABLE 7B Cut-off for positivity (e.i. sample is positive if more thanthis % of reads are meth- All 5 ylated at Up3

Up

Up35-1, Up3, Up3, Mark- the given Up3. Up3. Up3. Up3. Up3. Up

5-1, Up

5-2, Up35-1, Up35-1, Up35-2, Up

5-2, Up35-1, ers number Up35-1, Up35-1, Up35-2, Up35-2, Up10, Up10,Up10, Up35-2, Up35-2, Up10 Up10 Up10 to- of CpGs) Category Up10 Up27Up10 Up27 Up27 up27 up27 Up10 Up27 Up27 Up27 Up27 gether  1% Sensitiv-72% 69% 70% 67% 64% 77% 75% 72% 69% 74% 74% 77% 77% ity EAC  1%Sensitiv- 57% 50% 50% 36% 50% 57% 50% 57% 50% 50% 50% 57% 57% ity JCA 1% Sensitiv- 29% 38% 29% 38% 14% 29% 29% 29%

% 29% 29% 29% 29% ity LGD  1% Sensitiv- 89% 78% 89% 7

% 78%

9% 78% 89% 78% 89% 89% 89%

9% ity HGD  1% Speci- 97% 9

% 97% 9

% 97% 97% 97% 97% 98% 97% 97% 97% 97% ficity normal GEJ  1% Specific-95% 90% 95% 90% 90% 95% 95% 95% 90% 90% 90% 90% 90% ity for non- dysolBE  1% Specific- 84% 81% 94% 91% 91% 82% 91% 84% 81% 91% 91% 81% 81% ityall BE without dys- plasia(in- cluding SSBE, and non- dys- plastic BE)10% Sensitiv- 66% 65% 57% 56% 49% 71% 63% 66% 65% 62% 62% 70% 70% ityEAC 10% Sensitiv- 43% 36% 29% 14% 36% 50% 36% 43% 36% 36% 36% 50% 50%ity JCA 10% Sensitiv- 29% 38% 29% 38% 14% 29% 29% 29% 38% 29% 29% 29%29% ity LGD 10% Sensitiv- 67% 78% 44% 56% 44% 78% 56% 67% 78% 56% 56%78% 78% ity HGD

indicates data missing or illegible when filed

Table 8 shows the performance of selected combinations of methylated DNAmarkers (Up3, Up10, Up27, Up35-1, Up35-2) plus testing for mutations inTP53 (p53) for detection of different sample types in the esophagealbrushings samples presented in Tables 6 and 7. Samples are scored asdetected if any member of the marker combination panel scores the sampleas methylated or if analysis for TP53 mutations scores the sample asTP53 mutant. Shown is the performance of the marker panel in whichsamples are scored as detected if any methylation marker is detected asmethylated at greater than or equal to 1% of DNA reads, or if TP53 isdetected as mutant at greater than or equal to 3% or at greater than orequal to 10% of the DNA sequence reads. Also shown is the performance ofthe marker panel in which samples are scored as detected if anymethylation marker is detected as methylated at greater than or equal to10% of DNA reads, or if TP53 is detected as mutant at greater than orequal to 3% or at greater than or equal to 10% of the DNA sequencereads. Marker combination with greater than 90% specificity in all BEwithout dysplasia are preferred yellow. Marker combinations thatadditionally show superior sensitivity for EAC are further preferred.Particularly preferred marker combinations are: Up35-2 methylation plusTP53 mutation; Up35-2 methylation plus Up3 methylation plus TP53mutation; Up10 methylation plus Up3 methylation plus TP53 mutation;Up35-2 methylation plus Up10 methylation plus TP53 mutation; Up10methylation plus Up27 methylation plus TP53 mutation; Up35-2 methylationplus Up3 methylation plus Up10 methylation plus TP53 mutation

TABLE 8A Methyl cut- off (samples are positive if more than this % ofreads are methylated Up3 Up3 at the Up3 and and required Up-3 Up35-1Up35-2 Up10 Up27 Up35-2 Up10 Up27 number of P53 and and and and and andand and CpGs) cut-off Category p53 p53 p53 p53 p53 p53 p53 p53  1%  1%Sensitivity EAC 81%  84%  86%  75%  80% 88% 85% 85%  1%  1% SensitivityJCA 50%  64%  50%  57%  57% 50% 57% 57%  1%  1% Sensitivity LGD 25%  38% 25%  14%  13% 38% 14% 25%  1%  1% Sensitivity HGD 78%  78%  70%  33% 50% 89% 78% 78%  1%  1% Specificity normal 98%  98%  98%  98% 100% 98%97% 98% GEJ  1%  1% Specificity for 95% 100% 100% 100%  95% 95% 95% 90%non-dysplastic BE  1%  1% Specificity all be 94%  82%  91%  97%  91% 91%94% 88% without dyslplasia (including SSBE, and non-dysplastic BE)  1% 3% Sensitivity EAC 77%  82%  84%  73%  78% 85% 83% 83%  1%  3%Sensitivity JCA 50%  64%  50%  57%  57% 50% 57% 57%  1%  3% SensitivityLGD 25%  38%  25%  14%  13% 38% 14% 25%  1%  3% Sensitivity HGD 78%  78% 70%  33%  50% 89% 78% 78% 10%  3% Sensitivity EAC 69%  82%  80%  71% 71% 79% 79% 75% 10%  3% Sensitivity JCA 50%  57%  50%  57%  50% 50% 57%50% 10%  3% Sensitivity LGD 25%  38%  25%  14%  13% 38% 14% 25% 10%  3%Sensitivity HGD 44%  78%  60%  33%  50% 56% 44% 56% 10% 10% SensitivityEAC 60%  73%  69%  60%  65% 71% 70% 69% 10% 10% Sensitivity JCA 29%  43% 29%  43%  29% 29% 43% 29% 10% 10% Sensitivity LGD 13%  38%  25%   0% 13% 38%  0% 25% 10% 10% Sensitivity HGD 44%  78%  60%  33%  50% 56% 44%56%

TABLE 8B Methyl cut-off (samples are positive if more than this % ofreads are Up35-1, Up3, methylated Up35-2 Up35-2 Up10 Up3. Up3. Up3.Up35-2, Up35-2, Up35-2, at the and and and Up35-2, Up35-2, Up10, Up10,Up10 Up10 required Up10 Up27 Up27 Up10 Up27 Up27 up27 Up27 Up27 numberof P53 and and and and and and and and and CpGs) cut-off Category p53p53 p53 p53 p53 p53 p53 p53 p53  1%  1% Sensitivity EAC  92% 90% 85% 91%92% 87% 94% 94% 94%  1%  1% Sensitivity JCA  57% 57% 57% 57% 57% 57% 57%57% 57%  1%  1% Sensitivity LGD  29% 25% 14% 29% 38% 14% 29% 29% 29%  1% 1% Sensitivity HGD  67% 80% 44% 89% 89% 78% 78% 89% 89%  1%  1%Specificity normal  97% 98% 98% 97% 98% 97% 97% 97% 97% GEJ  1%  1%Specificity for 100% 95% 95% 95% 90% 90% 95% 90% 90% non-dysplastic BE 1%  1% Specificity all BE  91% 8

% 91% 91% 8

% 8

% 88% 88% 88% without dyslplasia (including SSBE, and non-dysplastic BE) 1%  3% Sensitivity EAC  90% 88% 83% 89% 90% 85% 92% 91% 91%  1%  3%Sensitivity JCA  57% 57% 57% 57% 57% 57% 57% 57% 57%  1%  3% SensitivityLGD  29% 25% 14% 29% 38% 14% 29% 29% 29%  1%  3% Sensitivity HGD  67%80% 44% 89% 89% 78% 78% 89% 89% 10%  3% Sensitivity EAC  85% 86% 77% 85%85% 81% 88% 87% 87% 10%  3% Sensitivity JCA  57% 50% 57% 57% 50% 57% 57%57% 57% 10%  3% Sensitivity LGD  29% 25% 14% 29% 38% 14% 29% 29% 29% 10% 3% Sensitivity HGD  56% 70% 44% 56% 67% 56% 67% 67% 67% 10% 10%Sensitivity EAC  77% 80% 71% 77% 79% 74% 81% 81% 81% 10% 10% SensitivityJCA  43% 29% 43% 43% 29% 43% 43% 43% 43% 10% 10% Sensitivity LGD  29%25% 14% 29% 38% 14% 29% 29% 29% 10% 10% Sensitivity HGD  56% 70% 44% 56%67% 56% 67% 67% 67%

indicates data missing or illegible when filed

Example 4: Analysis of Esophageal Cancer Informative Loci in FormalinFixed Paraffin Embedded Tissues

Additional studies were performed on DNAs extracted from formalin fixedparaffin embedded (FFPE) tissue samples of the stomach and esophagusthat capture different diagnostic categories other than Barrett's withhigh grade dysplasia and esophageal adenocarcinoma. Bisulfite convertedDNAs from each sample were amplified with bisulfite specific methylationindifferent primers corresponding to selected amplicons and theamplicons were then analyzed by bisulfite sequencing to determinemethylation status on the parental DNA templates.

Table 9 summarizes the side by side comparison of 8 methylated DNAmarkers in FFPE tissue samples of the stomach and esophagus that capturedifferent diagnostic categories other than Barrett's with high gradedysplasia and esophageal adenocarcinoma. Intestinal metaplasia isabbreviated as IM. Table 9 denotes for each marker the number ofmethylated cytosine bases required to be detected on a DNA sequence readto classify that read as methylated. Samples are detected as methylatedif greater than or equal to 1% of DNA sequence reads are classified asmethylated.

TABLE 9A Marker VIM SqBE5 SqBE Up7 SqBE11-2 CpG cut-off 6+ 13+ 23+ 5+Value (% positive samples or total number of samples sequenced) %positive Total number % positive Total number % positive Total number %positive Total number BE (IM) 90% 30 50% 12 50% 16  83% 30 GEJ/Cardiawith IM 82% 11  0%  5 33%  6 100% 11 GEJ/Cardia without  0% 58  0% 33 2% 47  31% 58 IM columnar mucosa 30% 10  0%  4 20%  5  30% 10 withoutIM taken from patients with concurrent IM at the same endoscopy NormalDistal  9% 23  8% 13  0% 16  13% 23 Esophagus-Squamous Squamous Mucosa 0% 10  0%  6  0%  5   0% 10 with REFLUX Esophagus Eosinophilic  0% 15 0%  5  0% 11   0% 15 Eosphagitis Gastic Mucosa with 22%  9  0%  2 20% 5  44%  9 IM Gastric Fundic  0% 24  0% 14  5% 21   8% 24 Mucosa withoutIM Helicobactor Pylori  8% 13 22%  9 13%  8  69% 13 Gastritis without IM

TABLE 9B Marker SqBE14-2 SqBE16 SqBE17 SqBE18 CpG cut-off 20+ 14+ 17+16+ Value (% positive samples or total number of samples sequenced) %Total % Total % Total % Total positive number positive number positivenumber positive number BE (IM) 50% 16 50% 26 69% 26 86% 29 GEJ/Cardia22%  9 18% 11 73% 11 70% 10 with IM GEJ/Cardia  2% 43  2% 55 7% 55  0%57 without IM columnar mucosa  0%  5 11%  9 11%  9 11%  9 without IMtaken from patients with concurrent IM at the same endoscopy NormalDistal 10% 20  5% 22  4% 23  4% 23 Esophagus- Squamous Squamous 17%  6 0%  8  0%  9  0% 10 Mucosa with REFLUX Esophagus Eosinophilic  0%  8 0% 12  8% 13  0% 12 Eosphagitis Gastic Mucosa  0%  5  0%  7 25%  8 14% 7 with IM Gastric Fundic  0% 14  0% 24  0% 24  8% 24 Mucosa without IMHelicobactor 14%  7  8% 13 46% 13 15% 13 Pylori Gastritis without IM

Table 10 summarizes the side by side comparison of 8 methylated DNAmarkers in FFPE tissue samples of the stomach and esophagus that capturedifferent diagnostic categories other than Barrett's with high gradedysplasia and esophageal adenocarcinoma. Intestinal metaplasia isabbreviated as IN. Table 10 denotes for each marker the number ofmethylated cytosine bases required to be detected on a DNA sequence readto classify that read as methylated. Samples are detected as methylatedif greater than or equal to 10% of DNA sequence reads are classified asmethylated.

TABLE 10A Marker VIM SqBE5 SqBE Up7 SqBE11-2 CpG cut-off 6+ 13+ 23+ 5+Value (% positive samples or total number of samples sequenced) % Total% Total % Total % Total positive number positive number positive numberpositive number BE (IM) 87% 30 50% 12 44% 16 70% 30 GEJ/Cardia 82% 11 0%  5 33%  6 91% 11 with IM GEJ/Cardia  0% 58  0% 33  2% 47 16% 58without IM columnar mucosa  0% 10  0%  4  0%  5 10% 10 without IM takenfrom patients with concurrent IM at the same endoscopy Normal Distal  9%23  8% 13  0% 16 13% 23 Esophagus- Squamous Squamous  0% 10  0%  6  0% 5  0% 10 Mucosa with REFLUX Esophagus Eosinophilic  0% 15  0%  5  0% 11 0% 15 Eosphagitis Gastic Mucosa 11%  9  0%  2 20%  5 22%  9 with IMGastric Fundic  0% 24  0% 14  5% 71  4% 24 Mucosa without IMHelicobactor  8% 13 22%  9 13%  8 46% 13 Pylori Gastritis without IM

TABLE 10B Marker SqBE14-2 SqBE16 SqBE17 SqBE18 CpG cut-off 20+ 14+ 17+16+ Value (% positive samples or total number of samples sequenced) %Total % Total % Total % Total positive number positive number positivenumber positive number BE (IM) 50% 16 42% 26 62% 26 76% 29 GEJ/Cardia22%  9 18% 11 64% 11 70% 10 with IM GEJ/Cardia  2% 43  2% 55  0% 55  0%57 without IM columnar mucosa  0%  5  0%  9 11%  9 11%  9 without IMtaken from patients with concurrent IM at the same endoscopy NormalDistal 10% 20  5% 22  4% 23  4% 23 Esophagus- Squamous Squamous 17%  6 0%  8  0%  9  0% 10 Mucosa with REFLUX Esophagus Eosinophilic  0%  8 0% 12  0% 13  0% 12 Eosphagitis Gastic Mucosa  0%  5  0%  7 13%  8 14% 7 with IM Gastric Fundic  0% 14  0% 24  0% 24  0% 24 Mucosa without IMHelicobactor 14%  7  8% 13 46% 13  8% 13 Pylori Gastritis without IM

Table 11 summarizes performance of different panels comprised ofcombinations of methylated DNA markers in FFPE tissue samples of thestomach and esophagus that capture different diagnostic categories otherthan Barrett's with high grade dysplasia and esophageal adenocarcinoma.Intestinal metaplasia is abbreviated as IM. Tables 9 and 10 denote foreach marker the number of methylated cytosine bases required to bedetected on a DNA sequence read to classify that read as methylated.Samples are detected as methylated if greater than or equal to 1% of DNAsequence reads are classified as methylated for any member of the markerpanel.

TABLE 11A Marker VIM VIM VIM VIM VIM SqBE5 SqBE5 combinations SqBE5SqBE7 SqBE16 SqBE17 SqBE18 SqBE7 SqBE16 BE (IM) 92% 88% 92% 88% 97% 71%70% GEJ/Cardia 60% 83% 82% 91% 80% 50%  0% with IM GEJ/Cardia  0%  2% 2%  7%  0%  3%  3% without IM columnar mucosa 25% 40% 33% 33% 33% 25% 0% without IM taken from patients with concurrent IM at the sameendoscopy Normal Distal  8%  6%  9%  9%  9%  8%  8% Esophagus SquamousSquamous  0%  0%  0%  0%  0%  0%  0% Mucosa with REFLUX EsophagusEosinophilic  0%  0%  0%  8%  0%  0%  0% Eosphagitis Gastic Mucosa  0%40% 14% 38% 43%  0%  0% with IM Gastric Fundic  0%  5%  0%  0%  8%  7% 0% Mucosa without IM Helicobactor 33% 13% 15% 46% 15% 29% 22% PyloriGastritis without IM

TABLE 11B Marker SqBE5 SqBE5 SqBE7 SqBE7 SqBE7 SqBE16 SqBE16combinations SqBE17 SqBE18 SqBE16 SqBE17 SqBE18 SqBE17 SqBE18 BE (IM)80% 91% 57% 79% 94% 78% 92% GEJ/Cardia 80% 40% 33% 50% 83% 73% 70% withIM GEJ/Cardia  9%  0%  2%  9%  2%  8%  2% without IM columnar mucosa 25% 0% 40% 20% 20% 22% 22% without IM taken from patients with concurrentIM at the same endoscopy Normal Distal  8%  8%  6%  0%  6%  9%  5%Esophagus- Squamous Squamous  0%  0%  0%  0%  0%  0%  0% Mucosa withREFLUX Esophagus Eosinophilic 20%  0%  0% 10%  0% 10%  0% EosphagitisGastic Mucosa 50%  0% 25% 40% 20% 33% 17% with IM Gastric Fundic  0%  7% 5%  5%  5%  0%  8% Mucosa without IM Helicobactor 44% 44% 25% 50% 25%46% 23% Pylori Gastritis without IM

Table 12 summarizes performance of different panels comprised ofcombinations of methylated DNA markers in FFPE tissue samples of thestomach and esophagus that capture different diagnostic categories otherthan Barrett's with high grade dysplasia and esophageal adenocarcinoma.Intestinal metaplasia is abbreviated as IM. Tables 9 and 10 denote foreach marker the number of methylated cytosine bases required to bedetected on a DNA sequence read to classify that read as methylated.Samples are detected as methylated if greater than or equal to 10% ofDNA sequence reads are classified as methylated for any member of themarker panel.

TABLE 12A Marker VIM VIM VIM VIM VIM SqBE5 SqBE5 combinations SqBE5SqBE7 SqBE16 SqBE17 SqBE18 SqBE7 SqBE16 BE (IM) 92% 81% 88% 85% 90% 71%60% GEJ/Cardia 60% 83% 82% 91% 80% 50%  0% with IM GEJ/Cardia  0%  2% 2%  0%  0%  3%  3% without IM columnar mucosa  0%  0%  0% 11% 11%  0% 0% without IM taken from patients with concurrent IM at the sameendoscopy Normal Distal  8%  6%  9%  9%  9%  8%  8% Esophagus- SquamousSquamous  0%  0%  0%  0%  0%  0%  0% Mucosa with REFLUX EsophagusEosinophilic  0%  0%  0%  0%  0%  0%  0% Eosphagitis  0% 20% 14% 13% 29% 0%  0% Gastic Mucosa with IM Gastric Fundic  0%  5%  0%  0%  0%  7%  0%Mucosa without IM Helieobactor 33% 13% 15% 46% 15% 29% 22% PyloriGastritis without IM

TABLE 12B Marker SqBE5 SqBE5 SqBE7 SqBE7 SqBE7 SqBE16 SqBE16combinations SqBE17 SqBE18 SqBE16 SqBE17 SqBE18 SqBE17 SqBE18 BE (IM)70% 91% 57% 71% 81% 74% 81% GEJ/Cardia 80% 40% 33% 50% 83% 64% 70% withIM GEJ/Cardia  0%  0%  2%  2%  2%  2%  2% without FM columnar mucosa 25% 0%  0% 20%  0% 11% 11% without IM taken from patients with concurrentIM at the same endoscopy Normal Distal  8%  8%  6%  0%  6%  9%  5%Esophagus- Squamous Squamous  0%  0%  0%  0%  0%  0%  0% Mucosa withREFLUX Esophagus Eosinophilic  0%  0%  0%  0%  0%  0%  0% EosphagitisGastic Mucosa  0%  0% 25% 20% 20% 17% 17% with IM Gastric Fundic  0%  0% 5%  5%  5%  0%  0% Mucosa without IM Helicobactor 44% 33% 25% 50% 25%46% 15% Pylori Gastritis without IM

Methodologies

Somatic mutations in TP53 were detected using the following method. TP53exons 2-11 were amplified using a multiplexed series of primer pairscovering all coding sequences and splice junctions. The primerscontained additional 5′ end sequences that were then used for secondaryamplification that introduced barcode sequences and Illumina 15 and 17sequences into the final PCR products. PCR products were mixed, purifiedand analyzed on an Illumina MiSeq instrument. Data analysis wasperformed using CLCBio software (Qiagen) and VariantStudio software(Illumina).

1. A method of diagnosing whether a subject has an esophageal neoplasiaor metaplasia, comprising: obtaining a sample from a subject; measuringthe amount of methylated cytosines in CpG dinucleotides in a vimentinnucleic acid sequence, or portion thereof, obtained from the sample;wherein if at least 80% of the cytosines in CpG dinucleotides in thevimentin nucleic acid sequence, or portion thereof, are methylated, thenthe vimentin nucleic acid sequence, or portion thereof, is considered amethylated read; and measuring the number of methylated reads present inthe sample; wherein if at least 1% of the vimentin nucleic acidsequences, or portions thereof, in the sample are methylated reads, thenthe subject is determined to have an esophageal neoplasia or metaplasia.2. The method of claim 1, wherein the vimentin nucleic acid sequencesfrom the sample are treated with bisulfite.
 3. The method of claim 2,wherein the sequence of the bisulfite converted nucleic acid sequencesis determined by next-generation sequencing.
 4. The method of claim 1,wherein the level of methylated cytosines is determined in an amplifiedportion of the vimentin nucleic acid sequence obtained from the subject.5. The method of claim 4, wherein between the amplification primers theamplified portion comprises 10 dinucleotides that correspond to or arederived from 10 CpG dinucleotides present in the native non-bisulfitetreated vimentin genomic sequence.
 6. The method of claim 5, wherein theprimers used to amplify the portion of the vimentin nucleic acidsequence comprise SEQ ID NOs: 16209 and
 16210. 7. The method of claim 4,wherein the amplified portion comprises the nucleotide sequence of SEQID NOs: 16207 and/or
 16208. 8. The method of claim 1, wherein if atleast 1.05% of the vimentin nucleic acid sequences, or portions thereof,in the sample are methylated reads, then the subject is determined tohave an esophageal neoplasia or metaplasia.
 9. The method of claim 1,wherein if at least 3% of the vimentin nucleic acid sequences, orportions thereof, in the sample are methylated reads, then the subjectis determined to have an esophageal neoplasia or metaplasia.
 10. Themethod of claim 1, wherein if at least 5% of the vimentin nucleic acidsequences, or portions thereof, in the sample are methylated reads, thenthe subject is determined to have an esophageal neoplasia or metaplasia.11. The method of claim 1, wherein if the subject is determined to havean esophageal neoplasia or metaplasia, then the method further comprisesadministering to the subject cryotherpy, photodynamic therapy (PDT);radiofrequency ablation (RFA); laser ablation; argon plasma coagulation(APC); electrocoagulation (electrofulguration); esophageal stent,surgery, and/or a therapeutic agent.
 12. A method of treating a subjecthaving an esophageal neoplasia or metaplasia, wherein it has beenpreviously determined that at least 1% of the vimentin nucleic acidsequences, or portions thereof, in a sample from the subject have atleast 80% of the CpG dinucleotides methylated, wherein the methodcomprises administering to the subject cryotherpy, photodynamic therapy(PDT); radiofrequency ablation (RFA); laser ablation; argon plasmacoagulation (APC); electrocoagulation (electrofulguration); esophagealstent, surgery, and/or a therapeutic agent.
 13. The method of claim 12,wherein the therapeutic agent is a proton pump inhibitor, a Histamine H2receptor blocking agents, an anti-reflux medication, a drug that movesfood thru the gastrointestinal tract more quickly, carboplatin andpaclitaxel (Taxol®); cisplatin and 5-fluorouracil (5-FU); ECF:epirubicine (Ellence®), cisplatin, and 5-FU; DCF: docetaxel (Taxotere®),cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda®); oxaliplatinand either 5-FU or capecitabine; doxorubicin (Adriamycin®), bleomycin,mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan, andirinotecan (Camptosar®), trastuzumab, and/or ramucirumab.
 14. The methodof claim 12, wherein the surgery is endoscopic mucosal resection (EMR),esophagectomy, and/or anti-reflux surgery.
 15. The method of claim 5,wherein the 10 CpGs correspond to those that, after bisulfite treatment,are included in SEQ ID Nos: 16211 and
 16212. 16-36. (canceled)
 37. Amethod of diagnosing whether a subject has an esophageal neoplasia ormetaplasia, comprising: obtaining a sample from a subject by means of abrushing; measuring the amount of methylated cytosines in CpGdinucleotides in a vimentin nucleic acid sequence, or portion thereof,obtained from the sample; wherein if at least 80% of the cytosines inCpG dinucleotides in the vimentin nucleic acid sequence, or portionthereof, are methylated, then the vimentin nucleic acid sequence, orportion thereof, is considered a vimentin methylated read; measuring theamount of methylated cytosines in CpG dinucleotides in an SqBE18 nucleicacid sequence, or portion thereof, obtained from the sample; wherein ifat least 70% or 75% of the cytosines in CpG dinucleotides in the SqBE18nucleic acid sequence, or portion thereof, are methylated, then theSqBE18 nucleic acid sequence, or portion thereof, is considered anSqBE18 methylated read; and measuring the number of methylated readspresent in the sample; wherein if at least 1% of the vimentin nucleicacid sequences, or portions thereof, in the sample are vimentinmethylated reads, and wherein if at least 3% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are SqBE18 methylatedreads, then the subject is determined to have an esophageal neoplasia ormetaplasia.
 38. The method of claim 37, wherein the vimentin and SqBE18nucleic acid sequences from the sample are treated with bisulfite. 39.The method of claim 38, wherein the sequence of the bisulfite convertednucleic acid sequences is determined by next-generation sequencing. 40.The method of claim 37, wherein the level of methylated cytosines isdetermined in an amplified portion of the vimentin nucleic acid sequenceand in an amplified portion of the SqBE18 nucleic acid sequence obtainedfrom the subject.
 41. The method of claim 40, wherein the amplifiedportion of the SqBE18 nucleic acid sequence comprises 21 dinucleotidesthat correspond to or are derived from 21 CpG dinucleotides present inthe native non-bisulfite treated SqBE18 genomic sequence.
 42. The methodof claim 40 or 41, wherein the amplified portion of the vimentin nucleicacid sequence comprises 10 dinucleotides that correspond to or arederived from 10 CpG dinucleotides present in the native non-bisulfitetreated vimentin genomic sequence.
 43. The method of claim 37, whereinif at least 1% of the vimentin nucleic acid sequences, or portionsthereof, in the sample are vimentin methylated reads, wherein if atleast 3.11% of the SqBE18 nucleic acid sequences, or portions thereof,in the sample are methylated reads, then the subject is determined tohave an esophageal neoplasia or metaplasia.
 44. The method of claim 37,wherein if the subject is determined to have an esophageal neoplasia ormetaplasia, then the method further comprises administering to thesubject cryotherpy, photodynamic therapy (PDT); radiofrequency ablation(RFA); laser ablation; argon plasma coagulation (APC);electrocoagulation (electrofulguration); esophageal stent, surgery,and/or a therapeutic agent.
 45. A method of treating a subject having anesophageal neoplasia or metaplasia, wherein it has been previouslydetermined that at least 1% of the vimentin nucleic acid sequences, orportions thereof, in a brushing sample from the subject have at least80% of the CpG dinucleotides methylated, wherein it has been previouslydetermined that at least 3% of the SqBE18 nucleic acid sequences, orportions thereof, in a brushing sample from the subject have at least75% of the CpG dinucleotides methylated, and wherein the methodcomprises administering to the subject cryotherpy, photodynamic therapy(PDT); radiofrequency ablation (RFA); laser ablation; argon plasmacoagulation (APC); electrocoagulation (electrofulguration); esophagealstent, surgery, and/or a therapeutic agent.
 46. The method of claim 45,wherein the therapeutic agent is a proton pump inhibitor, a Histamine H2receptor blocking agents, an anti-reflux medication, a drug that movesfood thru the gastrointestinal tract more quickly, carboplatin andpaclitaxel (Taxol®); cisplatin and 5-fluorouracil (5-FU); ECF:epirubicine (Ellence®), cisplatin, and 5-FU; DCF: docetaxel (Taxotere®),cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda®); oxaliplatinand either 5-FU or capecitabine; doxorubicin (Adriamycin®), bleomycin,mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan, andirinotecan (Camptosar®), trastuzumab, and/or ramucirumab.
 47. The methodof claim 45, wherein the surgery is endoscopic mucosal resection (EMR),esophagectomy, and/or anti-reflux surgery.
 48. A method of diagnosingwhether a subject has an esophageal neoplasia or metaplasia, comprising:obtaining a sample from a subject by means of a balloon; measuring theamount of methylated cytosines in CpG dinucleotides in a vimentinnucleic acid sequence, or portion thereof, obtained from the sample;wherein if at least 80% of the cytosines in CpG dinucleotides in thevimentin nucleic acid sequence, or portion thereof, are methylated, thenthe vimentin nucleic acid sequence, or portion thereof, is considered avimentin methylated read measuring the amount of methylated cytosines inCpG dinucleotides in an SqBE18 nucleic acid sequence, or portionthereof, obtained from the sample; wherein if at least 70% or at least75% of the cytosines in CpG dinucleotides in the SqBE18 nucleic acidsequence, or portion thereof, are methylated, then the SqBE18 nucleicacid sequence, or portion thereof, is considered a SqBE18 methylatedread; and measuring the number of methylated reads present in thesample; wherein if at least 0.95% of the vimentin nucleic acidsequences, or portions thereof, in the sample are vimentin methylatedreads, and wherein if at least 0.1% of the SqBE18 nucleic acidsequences, or portions thereof, in the sample are SqBE18 methylatedreads, then the subject is determined to have an esophageal neoplasia ormetaplasia.
 49. The method of claim 48, wherein the vimentin and SqBE18nucleic acid sequences from the sample are treated with bisulfite. 50.The method of claim 49, wherein the sequence of the bisulfite convertednucleic acid sequences is determined by next-generation sequencing. 51.The method of claim 48, wherein the level of methylated cytosines isdetermined in an amplified portion of the vimentin nucleic acid sequenceand in an amplified portion of the SqBE18 nucleic acid sequence obtainedfrom the subject.
 52. The method of claim 51, wherein the amplifiedportion comprises 21 dinucleotides that correspond to or are derivedfrom 21 CpG dinucleotides present in the native non-bisulfite treatedSqBE18 genomic sequence.
 53. The method of claim 51, wherein theamplified portion of the vimentin nucleic acid sequence comprises 10dinucleotides that correspond to or are derived from 10 CpGdinucleotides present in the native non-bisulfite treated vimentingenomic sequence.
 54. The method of claim 48, wherein if at least 1% ofthe vimentin nucleic acid sequences, or portions thereof and if at least0.76% of the SqBE18 nucleic acid sequences, or portions thereof, in thesample are methylated reads, then the subject is determined to have anesophageal neoplasia or metaplasia.
 55. The method of claim 48, whereinif at least 1% of the vimentin nucleic acid sequences, or portionsthereof and at least 1% of the SqBE18 nucleic acid sequences, orportions thereof, in the sample are methylated reads, then the subjectis determined to have an esophageal neoplasia or metaplasia.
 56. Themethod of claim 48, wherein if the subject is determined to have anesophageal neoplasia or metaplasia, then the method further comprisesadministering to the subject cryotherpy, photodynamic therapy (PDT);radiofrequency ablation (RFA); laser ablation; argon plasma coagulation(APC); electrocoagulation (electrofulguration); esophageal stent,surgery, and/or a therapeutic agent.
 57. (canceled)
 58. The method ofclaim 56, wherein the therapeutic agent is a proton pump inhibitor, aHistamine H2 receptor blocking agents, an anti-reflux medication, a drugthat moves food thru the gastrointestinal tract more quickly,carboplatin and paclitaxel (Taxol®); cisplatin and 5-fluorouracil(5-FU); ECF: epirubicine (Ellence®), cisplatin, and 5-FU; DCF: docetaxel(Taxotere®), cisplatin, and 5-FU; Cisplatin with capecitabine (Xeloda®);oxaliplatin and either 5-FU or capecitabine; doxorubicin (Adriamycin®),bleomycin, mitomycin, methotrexate, vinorelbine (Navelbine®), topotecan,and irinotecan (Camptosar®), trastuzumab, and/or ramucirumab.
 59. Themethod of claim 56, wherein the surgery is endoscopic mucosal resection(EMR), esophagectomy, and/or anti-reflux surgery. 60-64. (canceled)